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Presented  by 

*D R.  WILLIAM  J. 

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available  to  holders 
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in  Biological  Chemistry 


Columbia  Intonsttp^^ 

College  of  $f)P*tcians  anb  burgeons! 


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PHYSIOLOGICAL  CHEMISTRY 

LONG 


BY  THE  SAME  AUTHOR 


Elements  of  General  Chemistry 

Fourth  Edition.      33  Illustrations,     x-j-443 
pages.     Cloth.     $1.50  net. 

A  Text-Book  of 
Elementary  Analytical  Chemistry 

Third  Edition.      10  Illustrations,      x-f  297 
pages.     Cloth.     $1.25  net. 


P.  BLAKISTON'S  SON  &  CO. 

PHILADELPHIA 


A  TEXT-BOOK 


OF 


Physiological  Chemistry 


FOR 


STUDENTS  OF  MEDICINE 


JOHN    H.  LONG,  M.S.,  Sc.D. 

PROFESSOR   OF   CHEMISTRY   IN   NORTHWESTERN   UNIVERSITY   MEDICAL   SCHOOL,  CHICAGO 


SECOND   EDITION,  REVISED 
WITH  42  ILL  US  TEA  TIONS 


PHILADELPHIA 

P.    BLAKISTON'S   SON   &   CO. 

1012  Walnut  Street 

1909 


Copyright,  1909,  by  P.  Blakiston's  Son  &  Co. 


\^bs 


Press  of 

The  new  era  Printing  Company 

Lancaster,  pa. 


PREFACE  TO  THE  SECOND  EDITION. 


In  the  preparation  of  this  revision  a  number  of  important  changes 
have  been  made.  The  new  protein  classification  of  the  American 
societies  has  been  added,  while  a  few  points  based  on  older  notions 
of  protein  relations  have  been  dropped.  Additions  have  been  made 
in  several  other  chapters,  also,  and  most  frequently  in  the  general 
text. 

A  much  fuller  discussion  has  been  given  to  the  subject  of  the  urine, 
and  a  new  chapter  has  been  added  on  the  methods  of  urine  analysis. 
These  methods  embrace  not  only  the  usual  clinical  tests,  but  the  most 
important  quantitative  processes,  in  certain  directions,  as  well,  and 
are  given  in  sufficient  detail  for  practical  metabolism  work. 

I  have  endeavored  to  keep  the  book  within  reasonable  limits  as  to 
size  and  to  make  it  conform  to  the  needs  of  the  classes  of  students 
for  whom  it  is  written.  I  believe  it  covers  the  ground  which  should 
be  required  in  the  chemical  courses  in  medical  schools,  or  in  scientific 
schools  where  preparation  for  medicine  is  given.  Some  points  of 
minor  importance  for  beginners  are  printed  in  smaller  type,  but  in 
general  the  details  of  interest  to  specialists  only  are  omitted,  as  there 
is  danger  in  presenting  more  to  the  student  than  he  has  time  to 
properly  master.  This  danger  is  as  apparent  in  the  teaching  of 
physiological  chemistry  as  it  is  in  certain  other  lines  of  work. 

In  the  preparation  of  the  index  and  in  the  reading  of  the  proof  I 
have  been  greatly  aided  by  my  wife,  Catherine  Stoneman  Long,  and 
by  my  son,  Esmond  R.  Long,  to  both  of  whom  my  thanks  are  due. 

J.  H.  Long. 

Chicago,  July,  1909. 


FROM  THE  PREFACE  TO  THE  FIRST  EDITION. 


"  In  the  following  pages  I  have  attempted  to  present  a  brief  ac- 
count of  the  important  principles  of  physiological  chemistry  in  a  form 
suitable  for  the  use  of  medical  students  who  may  be  assumed  to  have 
completed  courses  in  the  elements  of  general  inorganic  and  organic 
chemistry.  From  the  very  necessities  of  the  case  a  work  of  this 
character,  dealing  with  many  topics  in  an  elementary  way,  must  be 
largely  a  compilation;  in  the  selection  of  material,  besides  consulting 
the  standard  hand  books,  I  have  made  free  use  of  the  recent  mono- 
graphs by  Cohnheim,  Effront  and  Oppenheimer,  as  well  as  of  numer- 
ous articles  in  the  Zeitschrift  fur  physiologische  Chemie,  the  Beitrage 
zur  chemischen  Physiologie  und  Pathologie  and  other  journals.  As 
the  book  is  intended  for  beginners  I  have  not  thought  it  necessary  to 
make  any  special  quotations  of  literature  references." 

"  A  considerable  number  of  illustrative  experiments  are  given  in 
the  text,  but  distinguished  by  being  printed  in  smaller  type.  These 
experiments  are  sufficiently  numerous  and  comprehensive  to  serve  the 
purpose  of  a  laboratory  course  parallel  with  the  general  course." 


vi 


Chapter 


Chapter 
Chapter 
Chapter 
Chapter 


TABLE  OF  CONTENTS. 


INTRODUCTION 
I.  Scope  and   Methods 


SECTION    I 
THE    NUTRIENTS 

II.  Inorganic  Elements.     Water.     Air.     Salts 7 

III.  The  Carbohydrates  and  Related  Bodies 17 

IV.  The  Fats  and  Substances  Related  to  Them 40 

V.  The  Protein  Substances 51 


SECTION    II 

FERMENTS    AND    DIGESTIVE    PROCESSES 

Chapter          VI.  Enzymes  and  Other  Ferments.     Digestion 96 

Chapter       VII.  Saliva  and  Salivary  Digestion 121 

Chapter      VIII.  The  Gastric  Juice  and  Changes  in  the  Stomach.  .  .  126 

Chapter         IX.  The  Products  of  Pancreatic  Digestion 144 

Chapter           X.  Changes  in  the  Intestines.     Feces 158 


SECTION    III 

THE   CHEMISTRY    OF   THE   BLOOD,   THE   TISSUES   AND 
SECRETIONS   OF   THE   BODY 

Chapter         XL  The  Blood 175 

Chapter        XII.  The  Optical  Properties  of  Blood.     The  Use  of  the 

Spectroscope  and  Other  Instruments 193 

Chapter      XIII.  Further  Physical  Methods  in  Blood  Examination. 
Freezing    Point    and    Electrical    Conductivity. 

The  Hematocrit 204 

Chapter      XIV.  Some  Special  Properties  of  Blood  Serum.     Bac- 
tericidal Action.     Precipitins,  Agglutinins,  Bac- 

teriolysins,  Hemolysins 216 

ptee        XV.  Transudations  Related  to  the  Blood 230 

"i  EB       XVI.   Milk   236 

vii 


viii  CONTENTS. 

Chapter    XVII.  The    Chemistry    of    the    Liver.     Bile.     Cells    in 

General 249 

Chapter  XVIII.  Chemistry  of   the   Pancreas    and   Other   Glands. 

Muscle,  Bone,  the  Hair  and  Other  Tissues 272 

SECTION   IV 

THE  END  PRODUCTS  OF  METABOLISM.   EXCRETIONS. 
ENERGY  BALANCE 

Chapter      XIX.  The  Excretion  of  Nitrogen,  Sulphur  and  Phos- 
phorus.    The  Urine 289 

Chapter        XX.  Some  Practical  Urine  Tests 313 

Chapter      XXI.  The   Gaseous   Excretion.     Respiration 356 

Chapter    XXII.  The  Energy  Equation 366 

Index 380 


PHYSIOLOGICAL    CHEMISTRY. 


INTRODUCTION. 

CHAPTER     I. 


Scope  and  Methods.  In  our  study  of  the  organized  world  the  most 
fundamental  problems  which  present  themselves  are  essentially  chem- 
ical. Beginning  with  the  mysterious  transformations  wrought  through 
the  energy  of  the  sun's  rays  in  such  simple  substances  as  the  carbon 
dioxide  and  aqueous  vapor  of  the  atmosphere,  when  these  bodies  come 
in  contact  within  certain  vegetable  cells,  and  following  the  history  of 
the  products  thus  formed  through  their  many  changes  in  the  plant 
organism  and  later  through  the  highly  complex  animal  structures, 
for  whose  formation  the  plant  cell  must  prepare  the  raw  material,  and 
finally  as  we  note  the  gradual  breaking  down  of  these  same  elaborate 
combinations,  with  liberation  of  energy  and  ultimate  restoration  of 
carbon  dioxide  and  water  and  nitrogen  to  the  air  and  soil  which  once 
had  held  them,  we  see  that,  step  by  step,  the  various  transformations 
which  occur  are  such  as  may  be  represented  by  the  equations  of  organic 
chemistry.  It  may  not  always  be  possible  to  express  these  equations  in 
simple  or  exact  form,  because  of  the  lack  of  knowledge  in  details, 
but  the  theoretical  feasibility  of  writing  such  expressions  we  every- 
where recognize. 

In  following  the  migrations  of  atoms  of  carbon,  hydrogen,  oxygen 
and  nitrogen  through  the  vegetable  and  animal  worlds,  our  inquiry 
naturally  widens  beyond  the  field  legitimately  claimed  by  chemistry. 
We  find  ourselves  at  the  very  outset  confronted  by  the  question  of  the 
final  forces  inaugurating  the  changes,  the  chemical  expression  of 
which  appears  often  so  extremely  simple.  It  may  be  the  part  of  wis- 
dom  to  admit  at  once  that  this  question  is  one  beyond  our  power  to 
answer.  Then,  again,  we  find  ourselves  attracted  by  questions  of 
form,  function  and  general  conditions  of  the  existence  of  organisms 
upon  the  earth,  in  addition  to  those  of  composition  and  mode  of  forma- 
tion.    In  touching  these  we  enter  upon  the  field  of  General  Biology 


2  PHYSIOLOGICAL    CHEMISTRY. 

and  soon  recognize  that  in  this  vast  and  independent  science  there  is 
much  contained  which  has  no  possible  bearing  on  our  problems  of 
chemistry.  But  some  knowledge  of  biological  science  is  certainly 
essential  to  a  proper  understanding  of  the  chemistry  of  living  beings. 

As  proper  subjects  of  inquiry  in  Physiological  Chemistry  we  recog- 
nize mainly  the  following:  (a)  The  nutrition  of  plants  and  animals 
and  the  composition  and  properties  of  the  nutrient  substances,  (b) 
The  changes  which  the  nutrients  undergo  before  and  during  the 
processes  of  assimilation,  (c)  The  agents  of  preparation  for  assimi- 
lation and  the  general  conditions  of  their  activity,  (d)  The  fate  of 
the  assimilated  nutrients  and  the  nature  of  the  products  of  degrada- 
tion.    (<?)  The  absorption  or  liberation  of  energy. 

In  the  broader  sense  the  discussion  is  extended  to  the  conditions 
appearing  in  the  life  history  of  plants,  and  the  lower  animals  as  well 
as  of  man,  but  ordinarily  the  narrower  field  of  the  life  of  man  and 
the  higher  animals  alone  is  considered,  and  in  the  present  work  the 
latter  limits  will  be  observed.  But  a  brief  discussion  of  the  general 
relations  of  plants  to  animals  will  not  be  out  of  place. 

At  one  time  it  was  very  generally  held  that  the  cell  activities  in  the 
plant  are  essentially  different  from  those  in  the  animal,  the  work  in 
the  one  case  being  looked  upon  as  wholly  synthetic,  while  in  the  other 
it  was  assumed  to  be  disintegration  or  analysis.  But  this  is  not  quite 
correct.  The  reactions  in  the  two  classes  of  structures  are  qualitatively 
much  the  same,  although  the  quantitative  differences  are  so  great 
that  we  almost  lose  sight  of  qualitative  similarities.  The  reactions  in 
the  plant  world  are  largely  endothermal  and  require  for  their  com- 
pletion the  constant  expenditure  of  external  energy.  This  energy  is 
derived  from  sunlight  and  through  its  agency  chlorophyll-bearing 
plants  are  able  to  effect  a  remarkable  condensation,  viz. :  that  of  carbon 
dioxide  with  water  accompanied  by  liberation  of  oxygen.  In  its 
simplest  terms  this  condensation  may  be  represented  as 

C02  +  H20  =  H2CO  +  02; 

that  is,  formaldehyde  and  oxygen  result. 

Formaldehyde  is  the  first  member  of  the  series  containing  the  mono- 
saccharoses  and  may  be  the  actual  starting  point  in  their  elaboration 
by  the  vegetable  cell.  Of  the  mechanism  of  further  transformations 
by  the  plant  we  know  but  little ;  it  is  likely  that  many  of  the  following 
changes  are  brought  about  by  the  action  of  soluble  ferments  or  en- 
zymes, which  will  be  referred  to  in  a  subsequent  chapter.  With  the 
completion  of  this  synthesis  a  large  amount  of  kinetic  energy  of  the 


INTRODUCTION.  3 

solar  rays  is  transformed  into  the  potential  energy  of  protein,  fat  or 
carbohydrate.  In  the  oxidation  of  the  plant  as  fuel  or  food  the  op- 
posite change  is  accomplished,  and  the  stored  up  energy  in  complex 
organic  molecules  is  liberated  as  heat,  electricity  or  muscular  motion. 
These  reactions  are  so  characteristic  for  plants  and  animals  that  we 
are  apt  to  lose  sight  of  others  which  also  take  place.  In  plants  there 
is  a  respiration  process  as  in  animals,  in  which  oxygen  is  absorbed  and 
carbon  dioxide  liberated,  and  in  the  dark  this  may  be  readily  observed, 
since  then  it  is  not  obscured  by  the  much  more  prominent  reduction 
process.  Indeed,  this  respiration  may  be  followed  in  the  light  in  the 
case  of  those  plants  which  are  free  from  chlorophyll.  Further  than 
this  there  are  parasitic  plants  which,  free  from  chlorophyll,  must  de- 
pend on  other  plants  for  their  nourishment;  they  consume  organic 
and  not  inorganic  materials,  and  in  this  behavior  resemble  animals 
completely. 

Then  it  must  be  remembered  that  the  activity  in  the  animal  is  not 
wholly  oxidation  or  degradation.  It  is  well  known  that  some  syn- 
theses are  constantly  taking  place  which  are  commonly  overlooked 
because  of  the  much  greater  importance  of  the  oxidation  reactions. 
In  recent  years  low  forms  of  animal  life  have  been  found  which 
contain  chlorophyll  grains  and  which  are  able  to  produce  oxygen  in 
presence  of  sunlight.  In  some  of  these  cases  the  chlorophyll  may  be 
present  in  a  symbiont  organism,  but  in  others  it  appears  to  be  diffuse, 
and  therefore  brings  the  animal  structure  containing  it  into  close  rela- 
tion with  vegetable  cells. 

In  the  essential  phenomena  of  life  plants  and  animals  have,  then, 
much  in  common ;  it  is  only  when  we  follow  them  into  details  that  the 
characteristic  differences  appear. 

From  the  nature  of  the  materials  entering  into  the  structure  of 
plants  and  animals  it  follows  that  the  discussions  of  physiological 
chemistry  are,  in  the  main,  but  special  cases  of  the  general  field  of 
organic  chemistry.  The  important  nutrients,  the  carbohydrates,  the 
fats  and  the  protein  substances,  are  all  organic  and  the  products  ap- 
pearing as  stages  in  their  metabolism  are  also  organic.  Therefore 
much  which  the  student  has  met  with  in  his  study  of  organic  chemistry 
may  be  found  repeated  in  his  work  in  physiological  chemistry.  Inas- 
much as  many  of  the  relations  to  be  now  traced  out  are  quantitative, 
it  is  highly  important,  also,  that  the  student  should  bring  to  the  work 
before  him  a  good  knowledge  of  the  principles  of  volumetric  analysis, 
as  these  will  be  applied  frequently  in  what  is  to  follow. 


4  PHYSIOLOGICAL    CHEMISTRY. 

Historical.  To  trace  the  beginnings  of  Physiological  Chemistry 
we  are  not  obliged  to  go  far  back  in  the  development  of  science. 
With  the  old  medical  chemistry  of  the  so-called  iatro  school  it  has 
nothing  in  common,  and  in  fact  is  in  no  sense  a  development  of  that 
science  of  the  sixteenth  and  seventeenth  centuries.  Before  the  days  of 
Lavoisier,  it  is  true,  some  little  advance  had  been  made  in  the  study 
of  bodies  of  animal  or  vegetable  origin,  but  without  a  rational  theory 
of  chemical  combination  the  isolated  facts  established  led  to  little  of 
real  value.  With  the  nature  of  respiration  explained,  however,  and 
the  identification  of  its  phenomena  with  other  phenomena  of  oxidation, 
the  way  was  opened  for  true  scientific  progress.  At  the  same  time 
accurate  methods  of  ultimate  organic  analysis  were  suggested  and 
soon  developed  by  the  followers  of  Lavoisier.  In  the  hands  of  Ber- 
zelius,  Gmelin  and  others  these  soon  began  to  furnish  results.  This 
brings  us  to  the  end  of  the  first  quarter  of  the  nineteenth  century,  a 
point  which  marks  the  real  beginning  of  our  science.  A  peculiar  dis- 
tinction between  inorganic  and  organic  bodies  had  gradually  arisen, 
and  had  come  to  be  commonly  accepted.  This  was  founded  on  the 
notion  that  while  the  former  might  be  produced  by  laboratory  proc- 
esses synthetically,  for  the  latter  group  nothing  similar  was  possible. 
By  this  arbitrary  limitation  research  was  naturally  greatly  curtailed. 
However,  in  1828,  Wohler  made  the  important  discovery  that  urea 
could  be  easily  formed  by  warming  a  solution  of  ammonium  cyanate, 
and  this  was  in  time  followed  by  others  of  equal  value,  pointing  to 
the  same  conclusion,  that  the  production  of  organic  compounds  is  in 
no  wise  dependent  on  the  aid  of  a  so-called  vital  force.  It  was  soon 
demonstrated  that  the  chemist's  laboratory,  no  less  than  nature's 
laboratory,  could  take  part  in  the  formation  of  these  substances,  and 
in  the  next  ten  years,  to  about  1840,  many  products  of  physiological 
interest  were  made.  The  great  Wohler  made  many  of  the  most  fruit- 
ful discoveries  of  this  epoch,  but  it  is  to  Liebig  that  we  owe  the  most. 
For  many  years  he  was  busily  engaged  in  perfecting  methods  of 
analysis,  and  with  these  developed  he  turned  his  attention  largely  to  the 
chemical  phenomena  of  vegetable  and  animal  life.  This  led  to  the 
publication,  in  1840,  of  his  epoch-marking  work,  "Organic  Chemistry 
in  Its  Relations  to  Agriculture  and  Physiology."  This  was  followed 
in  1842  by  a  work  giving  evidence  of  his  broadened  and  strengthened 
views,  "  Organic  Chemistry  in  Its  Relations  to  Physiology  and  Path- 
ology." These  works  passed  through  many  editions  and  were  trans- 
lated into  several  languages.  In  them  we  find  much  that  is  now 
considered  fundamental  in  physiology  and  physiological  chemistry, 
and  they  suggested  or  called  out  the  active  efforts  of  many  succeeding 


INTRODUCTION.  5 

investigators.  Not  a  few  of  these  men  are  still  living,  so  young  is 
our  science,  and  it  will  be  sufficient  to  merely  call  attention  to  the  names 
of  the  more  prominent  workers  who  followed  the  active  pioneers.  In 
Germany  C.  G.  Lehmann  made  many  important  contributions  to  the 
chemistry  of  the  blood  and  published  a  text-book  of  Physiological 
Chemistry  which  reached  a  third  edition  in  1853.  In  1858  F.  Hoppe- 
Seyler  published  the  first  edition  of  his  Physiological  and  Pathological 
Chemical  Analysis,  and  from  1877-81  his  Physiological  Chemistry 
in  four  parts  or  volumes.  This  work  contributed  greatly  to  our  sys- 
tematic knowledge.  C.  Voit,  since  1863  professor  of  physiology  in 
Munich,  began  his  valuable  studies  in  nutrition  and  metabolism  about 
1856,  and  continued  them  nearly  forty  years.  W.  Kiihne,  of  Heidel- 
berg, did  much  to  develop  the  chemistry  of  the  protein  substances, 
his  studies  dating  from  1859.  The  pupils  o-f  these  German  scholars 
are  to-day  among  the  most  active  investigators  in  all  fields  of  physi- 
ological chemistry. 

In  France,  Pasteur  must  be  mentioned  in  this  connection  on  account 
of  his  pioneer  investigations  on  fermentation  and  ferments,  a  subject 
of  far-reaching  importance.  CI.  Bernard  investigated  the  chemistry 
of  the  digestive  secretions  and  especially  the  behavior  of  sugar  in  the 
organism.  He  published  valuable  works  in  1853  and  1855,  which 
went  through  later  editions.  Somewhat  later  P.  Schuetzenberger,  in 
Paris,  made  important  additions  to  our  knowledge  of  the  chemistry 
of  the  protein  bodies  and  published  a  work  on  fermentation  which 
for  many  years  ranked  as  our  only  systematic  treatise  on  the  subject. 

At  the  present  time  physiological  chemistry  has  become  a  recog- 
nized department  of  study  in  the  United  States,  England  and  other 
continental  countries  as  well  as  in  Germany  and  France,  and  journals 
are  now  published  devoted  solely  to  its  interests.  The  rapidly  increas- 
ing number  of  investigations  published  in  these  journals  and  elsewhere 
attests  the  growing  importance  of  the  science  from  the  theoretical 
standpoint  as  well  as  in  its  practical  relations  to  medicine. 

It  remains  to  briefly  mention  the  development  of  another  field  of 
scientific  study  because  of  its  bearing  on  certain  problems  of  physi- 
ological chemistry.  In  the  last  quarter  of  the  eighteenth  century 
Lavoisier  clearly  showed  the  nature  of  combustion  and  the  relation  of 
animal  heat  to  respiration  and  the  oxidation  of  the  tissues.  Lavoisier 
and  Laplace  carried  out  the  first  quantitative  experiments  in  which  a 
c-ilorimeter  was  employed  to  measure  the  evolution  of  body  heat. 
These  were  repeated  by  Desprctz  in  T824  and  later  by  Dulong.  Since 
then  by  greatly  improved  methods  many  similar  investigations  have 
been  made. 


6  PHYSIOLOGICAL    CHEMISTRY. 

A  little  later  than  the  date  on  which  Lavoisier  and  Laplace  an- 
nounced their  important  researches  on  the  relation  of  animal  heat  to 
oxidation  of  foodstuffs,  Benjamin  Thompson,  Count  Rumford,  an- 
nounced a  discovery  of  equally  far-reaching  consequences.  He  made 
the  observation  that  the  heat  of  friction  between  two  pieces  of  metal 
may  be  absorbed  by  water  and  so  measured,  and  that  there  is  a  relation 
between  the  mechanical  work  lost  in  the  friction  and  the  heat  gen- 
erated. He  made  also  the  curious  observation  that  the  work  per- 
formed by  the  horse  in  one  of  his  experiments  in  which  friction  was 
produced  depended  in  turn  on  the  combustion  or  oxidation  of  the  food 
of  the  horse,  from  which  it  followed  that  indirectly  the  heating  of  the 
water  was  due  to  the  combustion  of  a  certain  amount  of  food.  But 
all  the  consequences  of  his  experiments  Rumford  did  not  see.  He  was 
mainly  interested  in  showing  the  absurdity  of  the  notion  of  the  ma- 
terial nature  of  heat,  then  commonly  held,  which  he  did  completely. 
It  remained  for  Joule  of  England  and  Mayer  of  Germany  to  point  out, 
nearly  fifty  years  later,  the  true  relation  between  heat  and  work.  In 
fine,  by  establishing  the  work  equivalent  of  heat  they  made  it  possible 
to  calculate  the  food  equivalent  of  work,  since  the  food  equivalent  of 
heat  had  been  already  proven.  These  relations  are  all  of  the  highest 
value  in  the  study  of  metabolism,  to  be  considered  in  the  sequel. 

The  discussions  of  the  earlier  part  of  the  eighteenth  century  placed 
in  clear  light  finally  the  full  meaning  of  the  doctrine  of  the  In- 
destructibility of  Matter.  These  later  discussions  developed  a  new 
doctrine,  that  of  the  Conservation  of  Energy,  the  recognition  of  which 
played  no  small  part  in  the  gradual  advance  of  physiological  as  well 
as  physical  science. 

It  is  the  intention  of  the  following  chapters  to  present  the  funda- 
mental facts  and  theories  of  physiological  chemistry  in  the  simplest 
possible  manner.  Much  matter  found  in  the  larger  hand-books  must 
necessarily,  therefore,  be  omitted  from  a  work  of  this  elementary 
character.  But  enough  will  be  given  to  furnish  the  student,  it  is 
hoped,  a  satisfactory  view  of  that  which  is  most  important  in  the 
science  at  the  present  time.  It  will  be  found  convenient  to  make  four 
general  divisions  of  the  subject,  as  follows : 

Section  I.     The  Nutrients  and  Related  Substances. 

Section  II.     Ferments  and  Digestive  Processes. 

Section  III.  The  Chemistry  of  the  Tissues  and  Secretions  of 
the  Body. 

Section  IV.  The  End  Products  of  Metabolism.  Excretions. 
Energy  Balance. 


SECTION    I. 

CHAPTER     II. 

THE    NUTRIENTS. 

INORGANIC  ELEMENTS.     WATER.    AIR.     SALTS. 

Composition  of  the  Body.  The  living  animal  body  is  composed 
in  the  mean  of  about  35  to  40  per  cent  of  solids  and  60  to  65  per  cent 
of  water.  In  adults  the  solids  are  somewhat  in  excess  of  this  amount, 
while  in  infants  they  are  lower,  perhaps  not  over  30  per  cent.  The 
elements  most  abundantly  present  are  carbon,  oxygen,  hydrogen,  nitro- 
gen, phosphorus,  sulphur,  chlorine,  potassium,  sodium,  calcium,  mag- 
nesium and  iron.  In  traces  only,  or  in  particular  tissues,  we  find 
iodine,  fluorine,  bromine,  silicon,  manganese,  copper  and  lithium. 
The  presence  of  these  in  minute  amount  seems  to  be  necessary  for 
the  existence  of  certain  animals.  These  elements  are  not  present  in 
the  free  state,  but  exist  combined  in  more  or  less  complex  compounds, 
the  degree  of  complexity  varying  between  that  illustrated  in  such 
simple  bodies  as  water  or  common  salt,  and  that  found  in  the  large 
protein  molecules  with  possibly  thousands  of  atoms  present. 

In  point  of  abundance  these  elements  are  found  in  the  body  in  about 
the  order  given  on  the  following  page. 

The  solids  of  the  body  are  both  organic  and  inorganic,  and  ap- 
proximately the  composition  of  the  whole  may  be  thus  represented : 

Per  Cent. 

Water  65 

Protein  substances  15 

Fats  14 

Other  organic  extractives 1 

.Mineral  matters   5 

It  must  be  remembered,  however,  that  the  fat  may  vary  widely  from 
the  above  number  and  therefore  change  the  ratio,  fat :  protein.  Among 
the  mineral  matters  calcium  phosphate  holds  the  first  place,  as  it 
makes  up  the  larger  part  of  bone  ash ;  carbonates  and  chlorides  of  the 
alkali  metals  make  up  the  remainder  largely. 

In  view  of  this  composition  of  the  body,  it  is  important  to  learn 
how  its  waste  is  replenished,  and  what  substances  must  be  or  may  be 

7 


PHYSIOLOGICAL    CHEMISTRY. 


Table  of  the  Elements  in  the  Body. 


Name  of  Element. 


Oxygen 
Carbon 
Hydrogen 
Nitrogen 

Calcium 

Phosphorus 

Potassium 

Sodium 

Chlorine 
Sulphur 


Magnesium 
Iron 


Iodine,  Fluorine, 
Silicon 


Per  Cent. 
Amount. 


66.0 
17-5 

10.2 
2.4 

1.6 
0.9 

0.4 

0.3 

0.3 
0.2 


0.05 
0.004 


traces. 


Occurrence. 


In  the  water  of  the  body,  in  the  fats,  the  protein  sub- 
stances and  in  nearly  all  the  tissues  and  salts. 

In  the  fats,  protein  substances  and  in  most  of  the  im- 
portant compounds  produced  in  the  body. 

In  water,  the  fats,  protein  substances  and  in  the  im- 
portant products   of  metabolism. 

Found  mainly  in  the  protein  substances  of  the  body. 
Also  in  many  of  the  metabolic  products  derived  from 
these. 

This  element  occurs  mainly  in  the  bones,  but  is  found 
in  the  blood  also  and  in  several  secretions  in  small 
amount. 

Is  found  principally  with  calcium  in  the  bones,  but 
occurs  also  in  several  complex  compounds  in  organic 
combination. 

Found  as  chloride,  carbonate  or  phosphate  in  many  of 
the  body  tissues  and  secretions.  Exists  also  in 
organic  combination. 

Occurs  combined,  as  does  potassium;  the  chloride  and 
carbonate  are  the  most  important  salts  and  are  found 
in  several  body  fluids. 

Found  in  combination  with  sodium  and  potassium,  also 
as  hydrochloric  acid  in  the  gastric  juice. 

This  element  is  important  as  occurring  in  the  protein 
compounds  of  the  body,  and  is  found  also  in  minute 
amount  in  other  combinations. 

Is  found  mainly  as  phosphate  and  carbonate  in  the  bones. 

Iron  occurs  in  the  important  hemoglobin  of  the  blood, 
as  an  integral  part  of  the  complex  molecule.  It  is 
found  also  in  inorganic  compounds  in  traces. 

Fluorine  is  found  in  the  teeth,  iodine  in  the  thyroid 
gland,  silicon  in  the  hair.  Besides  these,  other  ele- 
ments have  been  found  occasionally,  but  do  not  appear 
to  be  necessary. 


consumed  to  repair  the  constant  losses  and  enable  the  body  to  do  its 
proper  work.  This  leads  to  the  question  of  foods  or  nutrients  in  the 
broad  sense.  Beginning  with  the  inorganic  materials  used  by  the  body, 
we  have  first : 

•  Water.  As  it  appears  on  the  surface  of  the  earth  water  is  classed 
conveniently  as  hard  and  soft.  The  descending  rain,  after  the  dust  is 
washed  from  the  air,  consists  of  nearly  chemically  pure  water.  It 
holds  no  mineral  matters  dissolved,  and  is  contaminated  mainly  with 
a  small  amount  of  dissolved  carbon  dioxide.  Such  water  on  reaching 
the  earth  is  soft  and  can  replace  distilled  water  for  most  purposes. 
The  changes  which  follow  after  contact  with  the  soil  depend  on  the 
composition  of  the  latter.  If  the  strata  over  which  the  water  flows 
or  through  which  it  percolates  consist  of  sand,  quartz  or  silicate  rocks 
or  other  insoluble  materials  the  water  is  left  in  practically  pure  con- 
dition, and  is  the  water  usually  spoken  of  as  soft  water.  But,  on  the 
other  hand,  if  the  rain  water  comes  in  contact  with  limestone,  gypsum, 


THE    NUTRIENTS.  9 

or  other  slightly  soluble  substances,  something  goes  into  solution  and 
the  product  is  now  known  as  hard  water,  the  degree  of  "  hardness  " 
depending  on  the  amount  of  dissolved  solids.  Waters  containing  the 
carbonates  of  calcium  and  magnesium  are  described  as  temporarily  hard, 
since  the  carbonic  acid  which  holds  these  carbonates  in  solution  may 
be  removed  by  boiling,  which  causes  precipitation.  Calcium  sulphate 
or  chloride  in  water  can  not  be  precipitated  by  boiling  and  the  presence 
of  these  and  a  few  other  substances  produces  permanent  hardness. 

Moderate  amounts  of  these  mineral  matters  in  water  are  not  ob- 
jectionable; in  fact  waters  with  some  lime  and  magnesia  are  preferable 
to  absolutely  soft  water  for  drinking  purposes.  But  along  with  the 
inorganic  substances  the  water  may  take  other  things  from  the  soils 
with  which  it  comes  in  contact  that  are  not  so  desirable.  These  are 
various  partly  decomposed  organic  matters  of  animal  or  vegetable 
origin  and,  what  is  more  important,  minute  living  vegetable  cells, 
mostly  bacteria,  which  are  capable  of  causing  much  mischief  when 
taken  into  the  stomach  of  man.  It  is  very  generally  believed  that 
several  diseases  in  man  have  their  origin  in  the  consumption  of  water 
contaminated  in  this  way. 

Natural  Purification  of  Water.  But  it  must  not  be  supposed  that 
these  bacteria  are  always  harmful.  On  the  contrary  some  of  them 
are  the  common  agents  which  effect  the  natural  purification  of  waters 
containing  organic  matter,  in  which  they  incite  destructive  fermenta- 
tion or  putrefactive  changes  and  finally  oxidation.  As  a  result  of 
these  changes  harmless  inert  substances  such  as  nitrogen  or  nitrates, 
carbon  dioxide,  methane  and  water  are  produced  from  the  relatively 
complex  waste  or  excreta  of  the  higher  organisms.  When  we  speak 
of  the  spontaneous  or  self-purification  of  water  we  refer  to  a  series 
of  changes  in  which  these  bacteria  play  a  leading  part. 

Artificial  Purification  of  Water.  On  a  smaller  scale  water  may 
be  rendered  safe  and  suitable  for  household  use  by  several  methods. 
By  distillation  all  objectionable  matters  may  be  rejected  and  a  whole- 
some drinking  water  obtained.  It  is  possible,  also,  to  separate  prac- 
tically all  bacteria  and  other  solid  matters  by  filtration  through  beds 
of  fine  sand.  In  this  way  the  supplies  of  many  cities  are  obtained  at 
the  present  time.  Frequently  the  filtration  is  preceded  by  coagulation 
or  precipitation  of  the  organic  substances  by  means  of  some  suitable 
agent  such  as  alum  or  salts  of  iron,  or  lime. 

Tests  of  Drinking  Water.  In  the  sanitary  examination  of  water 
it  is  not  necessary  to  make  very  full  analyses  to  determine  its  value 
for  household  use.     A   few  tests  usually  suffice  to  discover  the  pres- 


IO  PHYSIOLOGICAL    CHEMISTRY. 

ence  or  absence  of  objectionable  substances.  For  example,  in  un- 
contaminated  waters  from  ordinary  springs,  lakes,  rivers  or  wells, 
chlorine  is  present  in  small  amount  only.  Any  excess  of  chlorine 
suggests  contact  with  sewage  or  household  waste  somewhere,  and  a 
quantitative  test  is  of  prime  importance  to  settle  this  point.  Such  a  test 
and  a  few  others  will  be  illustrated  below. 

Experiment.  The  Test  for  Chlorides.  A  test  is  often  made  in  this  way: 
Measure  out  200  cc.  of  the  water,  add  to  it  a  few  drops  of  a  solution  of  pure 
neutral  potassium  chromate,  and  then  from  a  burette  run  in,  with  constant  stirring, 
solution  of  tenth  normal  silver  nitrate  until  a  faint  reddish  precipitate  of  silver 
chromate  appears.  Each  cubic  centimeter  of  the  silver  solution  precipitates  3.54  mg. 
of  chlorine  from  common  salt  or  other  chloride,  and  when  the  last  trace  of  chlorine 
is  combined,  the  silver  begins  to  precipitate  the  chromate  with  production  of  red 
color.  The  chromate  acts  here  as  an  "  indicator,"  as  it  shows  just  when  the  chlorine 
is  all  combined  by  beginning  to  precipitate  itself. 

In  making  this  test  it  is  well  to  take  two  similar  beakers,  place  them  side  by  side 
on  white  paper,  pour  equal  amounts  of  water  in  each,  add  to  each  the  same  number 
of  drops  of  the  indicator,  and  then  with  one  make  the  actual  test  by  adding  the 
silver  solution.  Note  the  amount  used  to  give  a  light  shade  and  then  discharge  it  by 
adding  a  drop  of  salt  solution.  Now,  with  this  opalescent  or  turbid  liquid  for  com- 
parison add  silver  nitrate  to  the  second  beaker  until  the  light  yellowish  red  shade 
just  appears.    This  reading  is  usually  somewhat  more  accurate  than  the  first. 

As  a  result  of  the  decomposition  of  various  nitrogenous  matters 
ammonia  is  frequently  found  in  natural  waters.  Its  amount  is  there- 
fore a  measure  of  contamination  to  some  extent,  and  tests  for  its 
presence  are  always  made  in  sanitary  examinations.  In  practice  the 
test  is  usually  made  on  a  distillate  from  the  water  in  question,  but 
the  following  experiment  will  illustrate  the  behavior  of  the  reagent 
employed. 

Experiment.  Test  for  Ammonia.  Solutions  of  ammonia  or  ammonium  salts 
possess  the  peculiar  property  of  giving  a  yellowish  brown  color  with  what  is  known 
as  Nessler's  reagent  (a  solution  of  mercuric-potassium  iodide,  made  strongly  alka- 
line with  sodium  or  potassium  hydroxide).  With  more  than  traces  of  ammonia  a 
precipitate  is  formed. 

To  make  the  test  measure  out  50  cc.  of  the  water  in  a  large  test-tube,  or  tall 
narrow  beaker,  and  add  to  it  2  cc.  of  the  Nessler  solution.  By  placing  the  beaker 
on  a  sheet  of  white  paper  and  looking  down  through  it,  the  depth  of  color  can 
be  observed.  A  few  parts  of  ammonia  in  one  hundred  million  parts  of  water  can  be 
readily  seen  and  measured. 

The  Oxidation  Tests.  Pure  water  absorbs  free  oxygen  from 
the  atmosphere  but  has  no  tendency  to  decompose  compounds  to 
secure  it.  On  the  other  hand,  waters  containing  organic  matters  or 
certain  inorganic  contaminations  have  the  power  of  decomposing  oxy- 
gen salts  to  secure  the  oxygen  they  desire,  and  the  amount  of  oxygen  so 
taken  up  becomes  a  measure  of  the  impurity  of  the  water.     Potassium 


THE    NUTRIENTS.  I  I 

permanganate  is  a  salt,  which,  under  certain  conditions,  gives  up  its 
oxygen  to  waters  containing  organic  bodies  in  solution  and  is  fre- 
quently employed  in  water  analysis  for  this  purpose.  An  experiment 
will  show  one  way  in  which  it  is  used. 

Experiment.  Measure  out  about  ioo  cc.  of  pure,  carefully  distilled  water,  pour 
it  into  a  clean  beaker  in  which  water  has  just  been  boiled  and  add  5  cc.  of  pure 
dilute  sulphuric  acid  (1  to  3).  Place  the  beaker  on  wire  gauze  and  heat  to  boiling. 
Now  add  5  drops  of  a  dilute  permanganate  solution  (300  milligrams  to  the  liter) 
from  a  burette  or  dropping  tube  and  boil  five  minutes.     The  pink  color  persists. 

Repeat  the  experiment,  using  100  cc.  of  common  hydrant  water  to  which  a  trace 
of  egg  albumin  or  urea  has  been  added,  and  after  running  in  the  permanganate  boil 
again.  The  color  fades  out  and  more  may  be  added.  Finally,  after  sufficient  has 
been  added  the  pink  color  remains.  The  number  of  drops  or  cubic  centimeters  used 
is  a  measure  of  the  contamination  of  the  water,  although  often,  as  in  this  experi- 
ment, a  very  rough  one. 

The  Tests  for  Nitrites  and  Nitrates.  Nitrogenous  matters 
undergoing  oxidation  in  water  and  soil  usually  give  rise,  in  time,  to 
nitrites  and  finally  to  nitrates.  These  compounds  are  therefore  looked 
for  in  water  as  evidence  of  past  contamination.  In  most  instances 
nitrites,  as  a  less  advanced  stage  of  oxidation  than  nitrates,  suggest 
comparatively  recent  contamination.  The  tests  are  especially  inter- 
esting in  the  examination  of  well  and  spring  water. 

Chemists  are  acquainted  with  a  number  of  methods  for  the  detec- 
tion of  traces  of  nitrogen  in  the  form  of  nitrites  and  nitrates,  but  at 
the  present  time  certain  color  reactions  are,  because  of  their  simplicity, 
mainly  in  favor.    These  are  illustrated  by  the  following  tests : 

A  reagent  for  nitrites  is  prepared  by  dissolving  0.5  gm.  of  sul- 
phanilic  acid  in  150  cc.  of  acetic  acid  of  25tper  cent  strength,  and 
mixing  this  with  a  solution  of  0.1  gm.  of  pure  naphthylamine  in  200 
cc.  of  dilute  acetic  acid.  This  mixture  keeps  very  well  for  a  time  in 
the  dark. 

Experiment.  To  about  50  cc.  of  water  in  a  clean  beaker  add  2  cc.  of  the  above 
solution.  If  the  water  is  quite  free  from  nitrites  the  reagent  imparts  no  color  to 
it.  One  hundredth  of  a  milligram  of  nitrogen  as  nitrite  in  the  water  gives  a  faint 
pink  color  at  the  end  of  five  minutes ;  with  large  quantities  the  color  may  become 
deep  rose  red. 

Experiment.  A  nitrate  test  may  be  illustrated  in  this  manner :  To  the  residue 
obtained  by  evaporating  50  cc.  of  an  ordinary  river  or  lake  water  to  dryness  in  a 
porcelain  dish  add  1  cc.  of  phenolsulphonic  acid.  Rub  the  acid  over  the  bottom  of 
the  dish,  and  add  a  few  drops  of  dilute  sulphuric  acid.  Warm  the  dish  a  few 
minutes  and  add  25  cc.  of  water.  This  should  show  now  a  faint  yellow  color.  By 
supersaturating  with  ammonia  the  color  becomes  deeper.  In  this  experiment  picric 
acid  is  at  first  formed  if  a  nitrate  is  present  and  the  addition  of  ammonia  yields 
ammonium  picrate,  the  color  of  which  is  more  marked. 

For  the  interpretation  of  all  these  tests  works  on  sanitary  analysis  must  be 
consulted. 


12  PHYSIOLOGICAL    CHEMISTRY. 

Physiological  Importance  of  Water.  This  is  suggested  by  the 
large  proportion  in  which  it  is  present  in  the  animal  body,  as  shown 
above.  It  serves  primarily  as  the  general  solvent  for  all  the  solid 
foodstuffs  taken  into  the  system  and  assists  in  the  removal  of  the 
solid  waste  products  or  excreta.  To  accomplish  these  ends  it  must  be 
drunk  in  sufficient  quantity.  It  is  a  well  recognized  fact  that  most 
people  in  the  United  States  drink  too  little  water,  from  which  various 
ills  result.  Important  chemical  changes  within  the  body  are  dependent 
on  the  so-called  hydrolytic  action  of  water.  These  appear  mainly  in 
the  phenomena  of  digestion,  in  which  starches,  sugars,  protein  bodies 
and  fats  are  altered  before  absorption,  and  will  be  discussed  in  detail 
in  sections  to  follow. 

It  must  be  remembered  further  that  water  plays  a  very  important 
part  in  the  removal  of  heat  from  the  body.  For  each  gram  of  water 
evaporated  as  perspiration  or  in  the  breath  nearly  600  units  of  heat 
are  absorbed,  and  in  this  way  over  20  per  cent  of  the  heat  expenditure 
may  be  accounted  for. 

The  average  amounts  of  water  found  in  the  important  tissues  is 
shown  in  the  following  table : 

Per  Cent.  Per  Cent. 

Dentine    10.  Pancreas    78 

Fatty  tissues 20  Blood    79 

Bones 50  Kidney    83 

Elastic  tissue   50  Brain  (gray  matter)  ....  86 

Liver   •  • 70  Milk 


Skin 72  Vitreous  humor 98.5 

Muscles    75  Cerebro-spinal  fluid  ....  99.0 

Spleen 76  Saliva   99.5 

Air.  Besides  its  content  of  oxygen,  nitrogen  and  argon  the  at- 
mosphere contains  several  other  gases  in  small  amount.  The  most 
abundant  of  these  is  water  vapor,  with  smaller  traces  of  carbon  di- 
oxide, helium,  neon,  etc.  As  the  amount  of  aqueous  vapor  present  is 
extremely  variable  it  is  customary  to  give  the  analysis  of  the  dry  air 
only,  which  in  volume  per  cent  is  about  this,  the  rarer  gases  being 
included  with  the  nitrogen  and  the  argon : 

Per  Cent. 

Nitrogen     78.40 

Oxygen     20.94 

Argon 0.63 

Carbon  dioxide    03 

The  water  vapor  present  varies  with  the  temperature  and  other 
physical  conditions  and  may  sometimes  make  up  one  per  cent,  or  even 
more,  by  weight  of  the  whole  mass.  A  cubic  meter  of  fully  saturated 
air  contains  30.1  grams  of  aqueous  vapor  at  300  C,  and  75  per  cent  of 


THE    NUTRIENTS.  I  3 

this  is  frequently  present  in  the  hot,  "  close  "  weather  of  our  summers. 
It  is  this  high  proportion  of  moisture  which  renders  further  evapora- 
tion from  the  skin  so  difficult,  and  which  therefore  contributes  greatly 
to  our  bodily  discomfort. 

The  normal  carbon  dioxide  content  is  given  above  as  0.03  per  cent 
or  three  cubic  centimeters  in  ten  liters.  This  amount  is  greatly  ex- 
ceeded in  the  air  of  poorly  ventilated  houses,  but  is  not  in  itself  the 
cause  of  the  unpleasant  sensations  experienced  in  going  into  such  an 
atmosphere,  although  this  was  long  believed.  It  has  been  found  by 
experiment  that  one  can  breathe,  although  not  comfortably,  in  a  pure 
atmosphere  containing  as  much  as  3  per  cent  of  carbon  dioxide,  while 
an  atmosphere  contaminated  to  the  extent  of  1  per  cent  by  human 
respiration  would  be  practically  unbearable.  This  condition  is  doubt- 
less due  to  the  traces  of  organic  products  thrown  off  in  the  breath  and 
perspiration,  and  especially  to  the  decomposition  of  organic  matter 
on  the  unclean  skin.  The  carbon  dioxide  is  often  made  the  approxi- 
mate measure  of  the  contamination  of  inhabited  rooms,  because  of 
the  practical  difficulty  of  measuring  anything  else. 

In  respiration  the  air  is  modified  about  as  shown  by  these  figures: 

Inspired  Air  Expired  Air 

Per  Cent.  Per  Cent 

Nitrogen,  argon,  etc  79.0  80.0 

Oxygen    21.0  16.0 

Carbon    dioxide    03  4.0 

The  amount  of  oxygen  inhaled  each  day  by  a  full-grown  man  is 
not  far  from  500  liters,  while  the  volume  of  carbon  dioxide  exhaled 
is  somewhat  less,  about  450  liters  in  the  mean.  Later  something  will 
be  said  about  the  numerical  relation  existing  between  the  volume  of 
carbon  dioxide  eliminated  and  the  volume  of  oxygen  absorbed. 

The  most  accurate  method  of  finding  the  amounts  of  aqueous  vapor 
and  carbon  dioxide  in  the  air  is  to  aspirate  a  measured  volume  through 
a  series  of  weighed  absorption  tubes.  The  first  of  these  contain  dry 
granular  calcium  chloride  or  some  other  good  water  absorbent,  while 
the  following  tubes  contain  soda-lime  or  a  strong  potassium  hydroxide 
solution  to  absorb  the  carbon  dioxide.  The  increase  in  weight  of 
the  tubes  shows  the  amount  of  vapor  and  gas  absorbed  from  the  given 
volume  of  air.  For  quick  determinations  somewhat  less  exact  methods 
are  often  used  in  practice. 

The  atmosphere  often  contains  traces  of  other  gases,  as  ammonia, 
sulphurous  oxide,  oxides  of  nitrogen  and  ozone,  which  are  of  little 
physiological  importance  and  need  not  be  here  considered.  Of  greater 
importance  are  the  minute  organized   forms  everywhere  present  to 


14  PHYSIOLOGICAL    CHEMISTRY. 

some  extent,  at  least,  and  which  include  bacteria  and  many  other 
agents  of  putrefaction  and  fermentation.  Most  of  these  are  prac- 
tically harmless  in  respiration,  but  the  presence  of  others  is  an  element 
of  the  greatest  danger,  because  of  the  disturbances  they  occasion  when 
taken  into  the  body. 

MINERAL  SUBSTANCES  REQUIRED. 

Salts.  The  table  some  pages  back  gives  the  percentage  amount  of 
the  different  elements  which  make  up  the  human  body,  some  being 
united  in  organic  and  the  others  in  inorganic  compounds.  Aside  from 
water  the  most  abundant  and  important  of  the  inorganic  materials 
are  the  phosphates  and  carbonates  of  the  alkali-earth  metals  found  in 
the  bones,  the  alkali  chlorides  and  the  alkali  carbonates.  The  solid 
mineral  matter  or  ash  of  the  adult  body  amounts  in  the  mean  to  about 
5  per  cent;  not  far  from  four-fifths  of  this  content  comes  from  the 
skeleton,  while  about  one-tenth  of  it  is  derived  from  the  muscles. 
The  proportion  of  ash  in  the  different  tissues,  taken  in  the  moist 
condition,  is  approximately  as  follows : 

Per  Cent.  Per  Cent. 

Bones    33  Pancreas,  brain  1.0 

Cartilage    2  Lung,  heart    0.95 

Liver  and  spleen    1.5  Blood    0.93 

Muscles    1.3  Skin    0.75 

Kidney   1.2  Milk    0.70 

Leaving  traces  out  of  consideration,  it  appears  that  the  body  con- 
tains four  metallic  elements,  calcium,  sodium,  potassium  and  mag- 
nesium, which  exist  in  combination  with  four  acids,  viz.,  phosphoric, 
hydrochloric,  carbonic  and  sulphuric.  Of  all  these  compounds  the 
calcium  phosphate  of  the  bones  is  the  most  abundant. 

Phosphates.  The  phosphates  of  the  body  are  salts  of  the  common 
or  orthophosphoric  acid,  H3P04.  The  three  kinds  of  salts  possible 
here  are : 

Primary  phosphates,      MH2P04, 

Secondary  phosphates,  M2HP04, 

Tertiary  phosphates,       MsP04, 

The  alkali  salts  of  the  three  classes  are  readily  soluble  in  water. 
The  secondary  and  tertiary  phosphates  are  mostly  insoluble,  those 
of  the  alkali  metals  excepted.  Secondary  phosphates  are  converted 
into  pyrophosphates  by  heat  and  the  primary  phosphates  into  meta- 
phosphates.  To  most  indicators  the  primary  phosphates  show  acid 
behavior,  while  the  secondary  phosphates  are  feebly  alkaline.  The 
soluble  tertiary  phosphates  are  strongly  alkaline.  The  action  on 
different  indicators  must  be  remembered  in  attempting  to  estimate 
the  acidity  or  alkalinity  of  urine. 


THE    NUTRIENTS.  I  5 

The  phosphates  furnished  us  in  various  animal  and  vegetable  foods 
are  mainly  those  of  calcium  and  potassium,  but  it  is  likely  that  the 
larger  part  of  the  phosphorus  utilized  in  the  body  is  combined  in 
relatively  complex  organic  compounds,  the  lecithins  and  nucleins,  for 
example,  which  yield  phosphoric  acid  and  phosphate  in  the  final  oxi- 
dation. We  find  therefore  the  tertiary  calcium  and  magnesium  phos- 
phates, Ca3(P04)2  and  Mg3(P04)2  in  bones.  Acid  calcium  phos- 
phate of  the  formula  Ca(H2P04)2  occurs  in  some  of  the  body  fluids, 
and  is  an  important  urinary  excretion.  The  phosphate  CaHP04 
may  sometimes  be  deposited  from  the  urine.  Secondary  potassium 
phosphate,  K2HP04,  is  a  constituent  of  all  animal  cell  structures, 
possibly  in  soluble  form,  but  possibly,  also,  in  organic  combination. 
The  muscular  juice  is  rich  in  alkali  phosphates. 

Chlorides.  Chlorine  is  found  in  the  body  as  sodium  chloride  and 
potassium  chloride,  also  as  free  hydrochloric  acid  in  the  gastric  juice. 
In  our  foodstuffs  it  comes  to  us  mainly  as  sodium  chloride,  but  this 
by  double  decomposition  may  give  rise  to  the  potassium  chloride  later : 

K2COs  +  2NaCl  =  2KCI  +  Na2C03 

In  the  gastric  and  pancreatic  juices  and  in  the  blood  sodium  chloride 
is  more  abundant  than  potassium  chloride,  but  the  latter  is  in  excess 
in  the  cell  structures.  Chlorine  is  utilized  in  the  animal  body  only  as 
it  is  found  in  the  metallic  compounds  or  chlorides.  The  various 
organic  combinations  of  chlorine  can  not  replace  the  salts.  It  has 
been  pointed  out  by  Bunge  that  sodium  chloride  is  much  more  neces- 
sary in  the  food  of  man  or  animals  consuming  a  vegetable  diet  than 
it  is  when  the  diet  is  mainly  flesh. 

Carbonates.  The  carbonates  found  in  the  human  body  are  pro- 
duced there  from  the  carbonic  acid  of  oxidation.  Hard  waters  contain 
the  carbonates  of  calcium  and  magnesium,  but  these  must  suffer  de- 
composition when  taken  into  the  stomach,  and  the  traces  of  acid  gas  so 
expelled.  Besides  the  carbon  dioxide  of  tissue  oxidation  we  must 
consider  also  that  formed  by  several  ferment  processes  in  the  intestines. 
A  large  amount  of  the  gas  is  produced  in  this  way  and  part  of  this 
is  absorbed  into  the  circulation.  Under  certain  conditions  the  "  weak  " 
carbonic  acid  is  able  to  decompose  sodium  chloride  and  produce 
sodium  carbonate  and  free  hydrochloric  acid.  The  origin  of  the  latter 
in  the  gastric  juice  is  now  accounted  for  in  this  way,  while  the  sodium 
carbonate  formed  at  the  same  time  is  carried  into  the  blood  and 
through  this  to  other  parts  of  the  body,  where  other  carbonates  may 
be  made  by  double  decompositions.     The  soluble  alkali  carbonates  are 


1 6  PHYSIOLOGICAL    CHEMISTRY. 

the  most  abundant,  but  in  the  bones  and  in  the  teeth  calcium  carbonate 
forms  an  important  part.  Some  carbonates  are  always  excreted  by 
the  urine,  and  the  alkali-earth  carbonates  may  occasionally  appear  in 
the  sediment. 

Sulphates.  Sulphur  may  enter  the  body  in  a  variety  of  combina- 
tions but  nearly  all  of  it  is  finally  excreted  in  the  completely  oxidized 
form,  that  is,  as  sulphates,  by  the  urine.  Sulphur  in  organic  combina- 
tion is  found  in  all  protein  substances  and  in  the  final  oxidation  of 
these  compounds  in  the  body  sulphuric  acid  is  produced.  The  manner 
in  which  this  is  combined  before  excretion  will  be  discussed  later. 
The  amount  of  sulphur  normally  present  in  the  body  is  but  a  small 
fraction  of  one  per  cent  of  the  weight  of  the  latter  and  practically  all 
of  it  is  found  in  the  protein  or  protein-like  substances.  Among  these 
keratin  is  characterized  by  its  relatively  high  sulphur  content.  Of  the 
exact  manner  in  which  the  sulphur  is  combined  in  most  of  these  bodies 
but  little  is  known. 

Bases.  The  several  acid  radicals  referred  to  occur  in  combination 
with  metals  and  four  of  these  only  are  present  in  the  body  in  appre- 
ciable quantity.  These  are  calcium,  magnesium,  sodium  and  potas- 
sium, and  they  exist  in  the  form  of  salts  with  the  acid  radicals.  To 
these  four  the  iron  of  the  blood  must  be  added,  but  it  is  present  in 
organic  combination.  In  a  following  chapter  it  will  be  shown  to  what 
extent  these  bases  are  present  in  ordinary  foodstuffs.  Of  some  it  is 
important  that  they  enter  the  body  in  certain  forms  only,  otherwise 
their  utilization — assimilation — is  imperfect  or  even  impossible.  This 
is  especially  true  of  the  iron,  the  chief  use  of  which  is  in  the  building  up 
of  hemoglobin.  For  this  purpose  the  iron  of  the  mineral  salts  is  prob- 
ably not  available,  but  it  may  be  taken  from  certain  peculiar  organic 
combinations.  Many  mineral  matters  are  as  essential  for  the  growth 
of  the  body  as  are  the  organic  foods  to  be  described  in  the  next 
chapters  and  care  must  be  observed  to  provide  them  in  sufficient 
quantity,  especially  in  the  feeding  of  the  young.  There  is  some  reason 
for  believing  that  most  of  the  basic  material  assimilated  in  the  body 
is  in  the  form  of  complex  salts  or  organo-metallic  combinations  of 
some  kind.  The  sulphur,  phosphorus  and  carbon  are  so  found  and 
possibly  the  metals  also.  It  has  been  pointed  out  that  in  the  oxidation 
of  such  organo-metallic  compounds  carbonates  or  basic  salts  must 
result,  and  some  portion  at  least  of  these  salts  must  be  available  to 
combine  with  the  sulphuric  acid  arising  from  the  oxidation  of  the 
protein  substances.  According  to  Bunge  common  salt  is  the  only 
mineral  substance,  in  excess  of  that  furnished  by  the  usual  organic 
foods,  which  the  body  actually  demands  in  large  amount. 


CHAPTER     III. 

THE    CARBOHYDRATES    AND    RELATED    BODIES. 

Under  the  term  carbohydrate  it  has  long  been  customary  to  include 
a  number  of  bodies  with  closely  related  properties  and  similar  com- 
position, which  may  be  expressed  by  such  simple  formulas  as  C6H10O5, 
C0H12O6,  or  multiples  of  these.  The  term  carbohydrate  came  into 
use  long  before  the  structure  of  the  bodies  in  question  was  known. 
It  is  now  possible  to  describe  these  substances  in  their  relations  to  the 
fundamental  hydrocarbons  or  alcohols  and  this  classification  will  be 
therefore  briefly  explained. 

NATURE    OF    THE    CARBOHYDRATES. 

In  their  chemical  behavior  these  bodies  resemble  aldehydes  or  ke- 
tones in  certain  important  characteristics.  Like  the  latter  they  are 
often  strong  reducing  substances  and  most  of  them  form  combinations 
with  phenyl  hydrazine.  These  and  other  properties  suggest  that  they 
may  be  considered  as  aldehyde  or  ketone  derivatives  of  the  poly- 
hydric  alcohols,  which  relationship  is  shown  by  the  table  on  pages 
1 8  and  19,  which  contains  also  some  acid  derivatives  for  further 
illustration. 

Some  of  the  bodies  in  the  table  are  naturally  occurring  substances 
and  are  highly  important,  but  most  of  them  are  artificial.  The  aldo- 
hexoses  and  the  ketohexoses  are  closely  related  to  two  groups  of  more 
complex  bodies,  in  which  cane  sugar  and  starch  are  the  best  illustra- 
tions, and  with  them  form  the  important  class  of  carbohydrates  in 
the  more  restricted  sense. 

CARBOHYDRATES  PROPER. 

Following  the  usual  classification  we  have  then : 

Monoses,  or  monosaccharides, 
Saccharodioses,  or  disaccharides, 
Saccharotrioses,  or  trisaccharides, 
Polysaccharides. 

These  bodies  are  mostly  of  vegetable  origin,  but  some,  such  as 
sugar  of  milk,  are  found  in  the  animal  kingdom.  The  synthetic 
preparation  of  some  of  these  sugars  has  been  accomplished,  starting 
from  either  formaldehyde  or  the  mixture  called  above  glycerose. 
When  formaldehyde,  CH20,  is  treated  with  lime  or  other  weak 
3  17 


PHYSIOLOGICAL    CHEMISTRY. 


bases  it  polymerizes  or  condenses  to  a  mixture  of  sugars,  one  of  which 
has  been  isolated  in  pure  condition  and  is  known  as  a-acrose.  By  the 
condensation  of  the   mixture   of   glyceraldehyde   and   dioxy-acetone 


Relations  of  the  Carbohydrates. 


Polyhydric 
Alcohols. 

Aldehyde  Deriva- 
tives. 

Ketone     Deriva- 
tives. 

Acid  Derivatives. 

Acid  Derivatives. 

CH2OH 

CH2OH 
Glycol. 

CH.OH 

1 
CHO 

Glycol  aldehyde, 
diose. 

CH2OH 

!    " 

COOH 

Glycollic  acid 

COOH 

1 
COOH 

Oxalic  acid. 

CH2OH 

CHOH 

1 

CH2OH 
Glycerol. 

CH.OH 

1     " 
CHOH 

1 
CHO 

Glyceraldehyde. 

CH,OH 

1 
CO 

1 

CH2OH 
Dioxyacetone. 

CH.OH 

I 
CHOH 

1 
COOH 

Glyceric  acid. 

COOH 

1 
CHOH 

1 

COOH 
Tartronic  acid. 

V 

Glycerose  or  triose. 

CH2OH 

1 
CHOH 

1 
CHOH 

1 

CH2OH 
Erythrol. 

CH2OH 

CHOH 

1 
CHOH 

1 
CHO 

Aldotetrose. 

CH.OH 
I     " 
CHOH 

1 
CO 

1 

CH2OH 
Ketotetrose. 

CH.OH 

1 
CHOH 

1 
CHOH 

1 

COOH 
Erythritic  acid. 

COOH 

CHOH 

1 
CHOH 

1 
COOH 

Tartaric    acids. 

Erythrose. 

CH.OH 

1 
CHOH 

1 
CHOH 

1 
CHOH 

CH2OH 

Pentitols, 

arabitols,   etc. 

CH.OH 
1 
CHOH 

CHOH 

1 

CHOH 
1 

CHO 
Aldopentoses, 
arabinose,  etc. 

CH.OH 
1 
CHOH 

CHOH 

1 
CO 

1 

CH2OH 

Ketopentoses. 

CH2OH 

1 
CHOH 

1 
CHOH 

1 
CHOH 

COOH 

Tetrahydroxy- 

monocarboxylic 

acids,  arabonic 

acid,  etc. 

COOH 

1 

CHOH 
1 
CHOH 

1 
CHOH 

1 
COOH 

Trihydroxydi- 

carboxylic 

acids,  trioxyglu- 

taric  acids. 

V 

Pentoses. 

CH2OH 

CHOH 

1 
CHOH 

1 
CHOH 

1 
CHOH 

1 

CH2OH 

Hexitols, 

mannitol,  etc. 

CH.OH 

1 
CHOH 

1 
CHOH    ■ 

1 
CHOH 

CHOH 

CHO 
Aldohexoses, 
glucoses,  etc. 

CH.OH 

1     " 
CHOH 

1 
CHOH 

1 
CHOH 

1 
CO 

CH2OH 

Ketohexoses, 
fructose. 

CH.OH 

1 
CHOH 

1 
CHOH 

1 
CHOH 

CHOH 

1 

COOH 

Pentahydroxy- 

carboxylic  acids, 

mannonic   acids, 

dextronic   acid. 

COOH 

1 
CHOH 

1 
CHOH 

1 
CHOH 

1 
CHOH 

1 
COOH 

Tetrahydroxy- 

dicarboxylic 

acids,    saccharic 

acid,  etc. 

Y 

Hexoses. 

THE    CARBOHYDRATES    AND    RELATED    BODIES. 
Relations  of  the  Carbohydrates. — Continued. 


19 


Polyhydric  Aldehyde  Deriva-      Ketone  Deriva- 

Alcohols.  tives.  tives. 


C7H160T 


CSH1S0S 


CoHonOo 


C7H140T. 


Acid  Derivatives. 


^s-n-i6^'s 


^d-^-is^'9 


CH-OH 

(CHOH), 

COOH 

CHoOH 

(CHOH), 

COOH 

CH.OH 

(CHOH), 

COOH 


Acid  Derivatives. 


COOH 

(CHOH)5 

COOH 

COOH 

(CHOH)8 

COOH 

COOH 

(CHOH)r 
COOH 


(glycerose  or  triose)  mentioned  in  the  table  above  the  same  acrose 
has  been  obtained.  This  acrose  is  identical  with  the  sugar  mixture 
known  as  (d  -f-  /) -fructose. 

A  number  of  sugars  have  also  been  obtained  by  a  general  method  of  synthesis 
which  depends  on  the  fact  that  as  aldehydes  and  ketones  they  have  the  power  to 
unite  with  hydrocyanic  acid  and  produce  nitriles  of  acids  which  may  be  reduced 
to  new  aldehydes  with  a  larger  number  of  carbon  atoms  than  the  original  substance 
contained.  This  may  be  illustrated  by  starting  with  arabinose,  C5Hi0O5,  as  figured 
above.     This  with  hydrocyanic  acid  yields  a  cyanide  as  follows : 

CH2OH.(CHOH)3CHO  +  HCN  =  CH2OH.(CHOH)3.CHOHCN,       . 

and  this  by  the  usual  reaction  gives  arabinose  carboxylic  acid: 

CH2OH.(CHOH)3CHOHCN  +  2H20  =  CH2OH.(CHOH)3.CHOH.COOH  +  NH,. 

By  loss  of  water  from  this  acid  the  corresponding  lactone  is  formed: 

CH2OH.(CHOH)4COOH  — H20  = 

CH,OH.CHOH.CH.CHOH.CHOHCO  =  C0H10Oc. 


-0- 


By  reduction  with   sodium  amalgam  this  lactone  becomes  a  sugar,  identical  with 
that  obtained  by  the  other  condensation : 

CH.OH.CHOH.CH.CHOH.CHOH.CO  +  H,  = 

o ! 


CH2OH.(CHOH)4.CHO  =  QH]2O0. 

By  an  extension  of  the  principle,  sugars  with  7,  8  and  9  carbon  atoms  have  been 
obtained.  In  what  is  to  follow  a  brief  discussion  of  the  more  important  natural 
substances  will  be  given. 

THE  MONOSES  OR  MONOSACCHARIDES. 

A  number  of  pentose  and  hexose  bodies  must  be  considered  here. 

Pentoses.  Small  amounts  of  these  sugar-like  compounds  exist  in 
nature,  but  they  are  mostly  derived  from  simple  antecedent  substances 
called  pentosans.     The  pentoses  bear  the  same  relation  to  the  pentosans 


20  PHYSIOLOGICAL    CHEMISTRY. 

that  dextrose  bears  to  starch;  by  hydration  the  latter  compounds  are 
converted  into  the  former,  thus : 

C5Hs04  +  H20  =  C5H10O5. 

C6H10O5  +  H2Ot=C6H12O6. 

Among  the  pentoses  two,  known  as  /-arabinose  and  /-xylose,  are  the 
most  important. 

Arabinose,  or  pectin  sugar,  is  made  by  warming  cherry  gum, 
wheat  bran,  gum  arabic,  quince  or  gedda  mucilage,  exhausted  brewers' 
grains  and  various  other  substances  with  dilute  acids.  It  has  a  specific 
rotation  [a]D=  -f-  104.50.  When  boiled  with  dilute  hydrochloric 
acid  it  yields  furfuraldehyde. 

Xylose,  or  wood  sugar,  is  obtained  by  boiling  wood  gum  with 
dilute  sulphuric  acid.  Its  specific  rotation  is  -f-  19.40.  Like  the  pre- 
ceding body  it  is  a  reducing  sugar,  but  non-fermentable.  The  nutri- 
tive value  of  these  substances  for  man  is  low,  but  for  the  herbivora 
these  and  the  antecedent  pentosans  are  more  important,  as  they  appear 
to  be  rather  easily  digested  in  the  alimentary  tract  of  many  animals. 
Traces  of  pentoses  have  occasionally  been  found  in  human  urine. 

All  these  bodies  are  distinguished  from  the  sugars  proper  by  yield- 
ing relatively  large  quantities  of  furfuraldehyde  when  distilled  with 
hydrochloric  acid  or  sulphuric  acid,  which  reaction  may  be  illustrated 
by  the  following  test : 

Experiment.  In  a  small  retort  or  flask  fitted  with  a  delivery  tube  mix  5  gm.  of 
bran  and  100  cc.  of  ten  per  cent  hydrochloric  acid.  Connect  the  retort  or  flask  with 
a  condenser,  apply  heat  and  distil  over  about  half  the  liquid.  With  a  few  drops  of 
this  make  the  Schiff  furfuraldehyde  test.  Moisten  a  small  strip  of  paper  with 
aniline  acetate  obtained  by  mixing  equal  volumes  of  glacial  acetic  acid  and  aniline. 
Touch  this  test-paper  with  a  glass  rod  holding  a  drop  of  the  bran  distillate.  A 
bright  red  color  appears,  due  to  the  formation  of  furoaniline,  C4H3O.CH.  (C6H4NH2)2. 
The  reaction  is  extremely  delicate  and  serves  for  the  detection  of  traces  of  the 
products  yielding  furfuraldehyde,  C4H3O.CHO. 

The  Hexoses.  These  are  important  substances  represented  by  the 
general  formula  C6H1206.  Several  occur  widely  distributed  in  nature, 
being  found  in  ripe  fruits  and  elsewhere.  A  few  are  artificial  products 
formed  by  laboratory  operations.  Complex  combinations  of  these 
bodies  known  as  glucosides  are  also  common  and  are  essentially 
ethereal  salts  of  the  hexoses.  These  hexose  bodies  are  all  sweet, 
soluble  in  water,  nearly  insoluble  in  alcohol  and  are  all  reducing  sub- 
stances with  oxidizing  agents  like  Fehling's  solution.  They  undergo 
fermentation  readily,  and  all  the  important  forms  yield  alcohol  and 
carbon  dioxide  under  the  influence  of  the  yeast  organism.  Some  of 
them  yield  lactic  acid  when  acted  upon  by  the  proper  ferment.     By 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  21 

partial  oxidation  they  yield  monocarboxylic  acids,  such  as  mannonic 
acid  or  dextronic  acid,  and  by  the  more  pronounced  oxidation  they 
are  converted  into  dicarboxylic  acids,  as  saccharic  or  mucic  acid. 

A  reaction  of  great  importance  in  connection  with  the  hexoses  is 
that  which  they  exhibit  when  acted  upon  by  phenyl  hydrazine.  While 
this  is  a  general  aldehyde  or  ketone  reaction,  the  behavior  of  the 
hexoses  is  so  characteristic  as  to  require  mention.  With  one  molecule 
of  phenyl  hydrazine,  C6H5NH  —  NH2,  the  hexoses  yield  hydrazones, 
C6H1205  —  N  —  NH.C6H5,  the  ketone  or  aldehyde  oxygen  being  re- 
placed by  the  hydrazine  group.  The  hydrazones  are  mostly  soluble 
in  water.  An  excess  of  phenyl  hydrazine,  enough  to  give  two  mole- 
cules of  that  substance  to  one  of  the  hexose,  yields  bodies  called  osa- 
zones,  which  are  mostly  yellow,  insoluble  crystalline  compounds 
of  great  importance  for  the  separation  and  identification  of  sev- 
eral of  the  sugars.  They  are  represented  by  the  formula  C6H10O4 
(N  —  NH.C6H5)2,  and  by  warming  with  strong  hydrochloric  acid 
they  yield  peculiar  compounds  called  osones,  which  are  mixed  ketone 
and  aldehyde  structures.    These  reactions  will  all  be  illustrated  below. 

Glucose  (tf-glucose),  known  also  as  dextrose,  grape  sugar,  or 
diabetic  sugar,  is  the  best  known  representative  of  the  group.  It  is 
found  in  honey  and  many  fruit  juices,  often  associated  with  levulose 
or  fruit  sugar.  It  may  be  produced  by  the  action  of  weak  acids  on 
cellulose  or  starch  and  on  the  large  scale  is  so  made  from  the  latter 
substance.  Weak  sulphuric  acid  was  at  first  commonly  employed  and 
the  hydration  or  conversion  was  effected  under  pressure.  Hydro- 
chloric acid  is  now  generally  employed  in  making  a  commercial 
glucose,  the  acid  being  neutralized  with  sodium  carbonate  at  the  end 
of  the  reaction.  With  other  acids  the  action  is  much  slower  or  less 
complete.  In  the  section  below  on  the  behavior  of  starch  the  nature 
of  the  reaction  will  be  explained.  The  following  experiment  will 
illustrate  the  production  of  the  sugar  by  the  aid  of  sulphuric  acid : 

Experiment.  Make  a  paste  by  boiling  about  a  gram  of  starch  with  ioo  cc.  of 
water  in  a  glass  flask.  Add  10  drops  of  dilute  sulphuric  acid  (1:5)  and  boil  five 
to  ten  minutes.  Now  allow  the  liquid  to  cool,  remove  5  cc.  with  a  pipette,  dilute 
this  to  25  cc.  with  water  and  add  a  few  drops  of  iodine  solution;  a  blue  violet  color 
results,  showing  that  starch  or  a  starch-like  substance  is  still  present.  The  re- 
mainder of  the  acid  liquid  in  the  flask  is  next  boiled  steadily  for  one  hour,  a  little 
water  being  added  from  time  to  time  to  replace  that  lost  by  evaporation.  At  the 
end  of  an  hour  remove  5  cc,  dilute  and  test  with  iodine  solution  as  before.  The 
characteristic  starch  reaction  is  now  absent,  while  the  liquid  has  become  thin  and 
transparent. 

Neutralize  the  free  sulphuric  acid  by  addition  of  a  slight  excess  of  chalk  or  fine 
marble  dust,  heat  gently  to  complete  the  reaction.     Then  filter  and  evaporate  the 


22  PHYSIOLOGICAL    CHEMISTRY. 

filtrate  nearly  to  dryness  on  a  water-bath.  Allow  to  cool  and  notice  that  the 
residue  has  a  sweet  taste.  It  is,  in  fact,  glucose  and  the  experiment  illustrates  the  old 
method  of  manufacturing  glucose  on  the  large  scale.  Test  it  by  dissolving  a  little 
in  water  and  adding  a  few  drops  of  Fehling's  solution,  described  below.  On  boiling, 
a  yellowish  precipitate  appears  which  becomes  bright  yellow  and  finally  red. 

Under  certain  conditions  it  is  possible  to  obtain  a  pure  crystalline 
product  from  the  syrup  made  as  just  illustrated.  This  is  known  as 
crystallized  grape  sugar  or  pure  anhydrous  dextrose.  The  common 
commercial  product,  sold  as  glucose  syrup,  often  contains  much  un- 
converted dextrin  from  the  incomplete  hydrolysis  of  the  starch.  At 
the  present  time  large  quantities  of  glucose,  both  solid  and  liquid,  are 
made  and  used  in  the  fermentation  industries,  by  bakers  and  confec- 
tioners and  in  the  household.  Glucose  is  sweet,  but  not  as  sweet  as 
cane  sugar,  and,  because  of  the  fact  that  it  readily  undergoes  fermen- 
tation, it  can  not  replace  cane  sugar  for  certain  purposes,  such  as  the 
preparation  of  the  syrups  of  the  pharmacopoeia  or  the  canning  or 
preserving  of  fruits. 

The  typical  aldose  reactions  are  well  shown  with  a  solution  of  glu- 
cose. For  the  first  of  these  we  require  a  reagent,  referred  to  above 
as  Fehling's  solution,  which  is  made  as  follows : 

Fehling's  Solution.  Ex.  Dissolve  69.28  gm.  of  pure  crystallized  copper  sulphate 
in  distilled  water  and  dilute  to  make  a  liter  of  solution.  In  a  second  portion  of  dis- 
tilled water  dissolve  100  gm.  of  pure  solid  sodium  hydroxide  and  350  gm.  of  pure  re- 
crystallized  Rochelle  salt  by  aid  of  heat.  Cool  and  dilute  to  make  one  liter.  These 
solutions  when  mixed  yield  the  Fehling's  solution  proper.  It  is  best  to  mix  equal 
volumes,  quite  accurately  measured,  just  before  the  reagent  is  needed  for  use. 

This  Fehling's  solution  is  commonly  employed  as  a  qualitative  test 
for  reducing  sugars  in  general,  but  the  reaction  may  be  first  illus- 
trated by  a  simpler  method: 

Experiment.  Trommer's  Test.  Add  to  a  dilute  solution  of  glucose  a  consid- 
erable excess  of  strong  potassium  hydroxide  solution  and  then  a  very  few  drops 
of  a  dilute  copper  sulphate  solution.  This  produces  no  precipitation  but  imparts  a 
deep  blue  color.  On  warming  the  solution  a  yellowish  precipitate  forms,  which 
grows  bright  red  by  boiling.  This  is  cuprous  oxide,  and  the  test  is  known  as 
"  Trommer's"  test.  It  is  frequently  employed  to  detect  the  presence  of  sugar  in 
liquids,  especially  in  urine,  but  on  the  whole  is  not  as  satisfactory  as  the  next  one. 

Experiment.  Fehling's  Test.  To  a  very  weak  glucose  solution  add  an  equal 
volume  of  diluted  Fehling's  solution.  The  mixture  remains  deep  blue  in  the  cold, 
but  on  heating  a  yellowish  precipitate  turning  to  red  is  soon  produced.  This  pre- 
cipitate of  cuprous  oxide  comes  from  the  reduction  of  the  cupric  compound  held 
in  solution  in  the  test  reagent.  In  the  first  or  Trommer  test  the  first  indication  of 
the  presence  of  a  sugar  is  the  formation  of  a  deep  blue  clear  solution  rather  than 
a  greenish  blue  precipitate  of  cupric  hydroxide  which  would  result  from  the  action 
of  the  copper  sulphate  and  alkali  alone.  But  cupric  hydroxide  dissolves  in  solu- 
tions of  sugars  and  other  polyhydric  alcohols,  the  solution  being  deep  blue,  and 
stable  in  the  cold.     In  the  case  of  glucose  and  other  reducing  (aldehyde  or  ketone) 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  23 

sugars  this  stability  is  only  temporary,  since  reduction  follows  on  boiling.  If  the 
polyhydric  alcohol  employed  to  produce  the  deep  blue  solution  is  not  a  reducing 
substance  the  liquid  remains  clear  and  stable,  even  on  boiling.  This  is  the  case  with 
the  Fehling's  solution,  in  which  the  cupric  hydroxide  is  held  dissolved  through  the 
alcoholic  behavior  of  the  Rochelle  salt.  Similar  solutions  are  made  by  the  aid 
of  glycerol  (trihydric)  and  mannitol  (hexahydric).  The  Fehling  test  has  this  ad- 
vantage over  the  Trommer  test,  that  in  the  latter  if  too  much  copper  sulphate  is 
used,  and  little  sugar  is  present  the  precipitate  on  boiling  may  be  mainly  black 
cupric  oxide  instead  of  the  red  cuprous  oxide.  With  the  Fehling  liquid  no  black 
precipitate  can  form. 

Experiment.  Bismuth  Reduction  Test.  Add  to  a  glucose  solution  some  strong 
potassium  hydroxide  solution  and  then  a  very  small  amount  of  bismuth  subnitrate. 
For  an  ordinary  test  a  few  milligrams  will  be  enough.  On  boiling,  a  black  pre- 
cipitate appears,  which  frequently  forms  a  bright  mirror  on  the  walls  of  the  test- 
tube.  This  precipitate  seems  to  be  a  mixture  of  metallic  bismuth  with  some  oxide, 
and  shows  the  strong  reducing  power  of  the  sugar.  Similar  reductions  may  be 
obtained  from  alkaline  solutions  of  several  heavy  metals,  but  these  tests  illustrate 
the  general  principle. 

Another  test  which  serves  for  the  recognition  of  even  minute  traces 
of  glucose  and  other  sugars,  is  the  following,  proposed  by  Molisch: 

Experiment.  To  a  small  amount  of  a  dilute  sugar  solution  add  two  drops  of  a 
solution  of  a-naphthol,  containing  about  20  gm.  in  100  cc.  On  shaking  the  liquid 
becomes  turbid.  Now  add  to  it  an  equal,  or  slightly  greater,  volume  of  pure  strong 
sulphuric  acid  and  shake.  A  deep  violet  color  appears,  which  gives  place  to  a 
violet  precipitate  on  addition  of  water.  This  reaction  has  been  shown  to  be  due 
to  the  combination  of  the  a-naphthol  with  furfuraldehyde  produced  by  the  action 
of  sulphuric  acid  on  the  sugar  present. 

Experiment.  Phenyl  Hydrazine  Test.  A  characteristic  reaction  of  great  prac- 
tical value,  referred  to  above,  is  given  on  the  addition  of  phenyl  hydrazine  to  a 
solution  of  glucose  under  certain  definite  conditions. 

To  20  cc.  of  a  dilute  glucose  solution  add  about  a  gram  of  phenyl  hydrazine 
hydrochloride,  and  two  grams  of  sodium  acetate.  Heat  on  the  water-bath  half  an 
hour,  and  then  allow  the  liquid  to  cool.  There  will  now  be  found  a  beautiful 
yellow  crystalline  precipitate  of  phenyl  glucosazone,  the  nature  of  which  is  best- 
seen  under  the  microscope.  This  test  is  one  of  great  delicacy,  and  has  been  applied 
to  the  detection  of  traces  of  sugar  in  urine.  But  care  must  be  taken  to  keep  the 
reagent  in  considerable  excess,  since  otherwise  the  soluble  hydrazone  may  be  formed. 
The  melting  point  of  the  pure  osazone  is  205°  C.  The  following  reactions  illustrate 
the  combination  of  the  phenyl  hydrazine : 

C,HuO.  +  C6HB.NH.NH2  =  C.HuO^N.NH.C.H.  +  H20, 

C.HuO.  +  2C,H8NH.NH2  =  C6H]0O4.(N.NH.QHB)2  +  2H20  +  H2, 

Phenyl  glucosazone 

C.H..NH.NH,  +  H2  =  CeH8NH2  +  NH3. 

By  treatment  with  strong  hydrochloric  acid  the  osazone  decomposes  to  yield  an 
osone  and  phenyl  hydrazine  hydrochloride : 

CH1.04.(N.NH.C,H,)I  +  2HCI  +  2H20  = 

2C.H5.NH.NH2.HC1  +  CH20H.(CH0H)8.C0.CH0. 

Glucosone 


24  PHYSIOLOGICAL    CHEMISTRY. 

This  osone  on  reduction  with  nascent  hydrogen  yields  a  sugar,  not  glucose,  but 
d-fructose,  or  levulose: 

CH2OH.(CHOH)3.CO.CHO  +  H2  =  CH2OH(CHOH)3.CO.CH2OH. 

Glucose  and  levulose   (cf-fructose)   yield  the  same  glucosazone;   we  are  therefore 
able  by  this  reaction  to  pass  from  one  sugar  to  the  other. 

The  ready  fermentation  of  glucose  will  be  shown  later  in  the  dis- 
cussion of  fermentation  reactions  in  general.  The  production  of  glu- 
cose from  cane  sugar  will  also  be  explained.  The  specific  rotation 
of  glucose  in  20  per  cent  solution  is  given  by  the  formula  [a]z>  =  53°, 
and  increases  slightly  with  the  concentration. 

gJ-Fructose,  fruit  sugar,  levulose,  is  a  ketohexose  similar  to  d- 
glucose  in  some  respects  but  very  different  in  others.  It  occurs  in 
honey  and  sweet  fruits,  but  is  not  easily  separated  in  a  pure  state 
because  it  is  very  soluble  and  does  not  readily  crystallize.  The  prepa- 
ration of  glucose  from  ordinary  starch  has  been  referred  to  above. 
In  like  manner  fructose  may  be  obtained  from  certain  less  common 
starches,  especially  from  inulin  by  hydrolysis  with  very  weak  acid. 
In  pure  condition  this  sugar  has  no  technical  importance. 

The  various  reduction  and  fermentation  reactions  are  shown  by 
fructose  as  well  as  by  glucose,  but  the  quantitative  relation  between 
copper  hydroxide  and  fructose  is  not  quite  the  same  as  with  glucose. 
As  they  yield  the  same  osazone  the  phenyl  hydrazine  test  can  not  be 
employed  to  distinguish  between  them.  The  most  characteristic  prop- 
erty of  fructose  is  found  in  its  optical  behavior.  While  the  specific 
rotation  of  c?-glucose  is  about  53 °  to  the  right,  that  of  d-fructose  is, 
at  200  C,  and  for  a  strength  of  20  per  cent,  about  93 °  to  the  left. 
Because  of  this  behavior  the  sugar  is  commonly  called  levulose.  An- 
other reaction  which  may  be  applied  is  this : 

Experiment.  Dissolve  resorcin  in  20  per  cent  hydrochloric  acid  and  heat  a  little 
of  this  solution  with  the  levulose  solution  to  be  tested.  A  red  color  results.  At 
the  same  time  a  precipitate  forms  which  may  be  dissolved  in  alcohol  with  a  red 
color.     One-tenth  gram  of  resorcin  to  5  cc.  of  the  acid  is  sufficient. 

^-Galactose.  This  is  the  third  hexose  of  importance,  but  it  is  not 
a  natural  substance.  In  the  inversion  of  milk  sugar  by  weak  acids 
galactose  is  formed  along  with  glucose,  and  it  results  also  from  the 
action  of  acids  on  several  gums.  The  sugar  is  readily  soluble  in 
water,  fermentable  and  dextro-rotatory  like  glucose.  It  forms  a  char- 
acteristic osazone.  It  reduces  Fehling's  solution  but  not  in  the  same 
proportion  as  glucose.  On  reduction  it  yields  dulcitol,  which  shows 
its  chemical  relations  most  characteristically.  On  oxidation  it  yields 
galactonic  and  mucic  acids. 

J-Talose  is  an  unimportant  aldose  of  artificial  origin. 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  25 

Sorbinose  is  a  ketone  sugar  obtained  from  the  juice  of  the  moun- 
tain ash  berry.     It  is  levorotatory  and  non- fermentable  with  yeast. 

Invert  Sugar.  This  name  is  given  technically  to  the  mixture  of 
glucose  and  fructose,  equal  molecules,  produced  by  the  action  of  weak 
acids  on  cane  sugar,  as  described  below. 

THE  CANE  SUGAR  GROUP. 

The  Saccharobioses  or  Disaccharides.  These  are  sugars  of  the 
formula  C12H22011  and  are  important  substances.  The  best  known 
representatives  of  the  class  are  cane  sugar,  milk  sugar  and  malt  sugar, 
all  of  which  are  natural  products,  with  cane  sugar  the  most  abundant. 
These  bodies  are  closely  related  to  the  hexoses,  two  molecules  of  the 
latter  being  in  some  manner  condensed  or  united  to  produce  one  of  the 
former.  The  disaccharides,  on  the  other  hand,  by  treatment  with 
weak  acids  or  certain  ferments  break  up  easily  into  two  molecules  of 
a  hexose,  water  being  added  in  the  reaction : 

CJSJOa  +  H20  =  CHuO,  +  QH120,. 

The  hexose  molecules  formed  may  be  alike  or  different,  and  the 
process  of  converting  the  disaccharides  into  monosaccharides  in  this 
manner  is  called  "  inversion."  By  this  inversion  the  following  changes 
should  be  noted : 

Saccharose  or  cane  sugar  yields  glucose  and  fructose. 

Lactose  or  milk  sugar  yields  glucose  and  galactose. 

Maltose  or  malt  sugar  yields  glucose  and  glucose. 

Saccharose.  This  sugar  has  been  known  from  earliest  times  to 
some  peoples,  but  did  not  become  an  article  of  commerce  until  after 
the  discovery  of  the  Americas.  It  is  found  in  the  juices  of  various 
canes,  several  kinds  of  beets,  the  saps  of  many  trees  and  in  many  seeds 
and  nuts.  On  the  commercial  scale  it  is  produced  from  the  beet  and 
canes,  and  in  smaller  amount  from  maple  sap. 

Cane  sugar  does  not  undergo  fermentation  directly  with  pure  yeast, 
but  by  prolonged  action  of  common  yeast  on  a  dilute  solution  of  the 
sugar  fermentation  appears.  This  is  due  to  the  fact  that  the  crude 
yeast  contains  an  inverting  enzyme  known  as  invertase  which  produces 
glucose  and  fructose  which  then  yield  to  the  true  fermentation.  Cane 
sugar  gives  no  combination  with  phenyl  hydrazine,  and  is  not  a  re- 
ducing sugar.  These  facts  point  to  the  absence  of  aldehyde  or  ketone 
groups  in  the  large  molecule.     An  experiment  illustrates  this: 

Experiment.  Prepare  a  dilute  solution  of  pure  cane  sugar  and  boil  it  with 
Fehling  solution  in  the  usual  manner.  Observe  that  no  reduction  of  the  copper 
compound  takes  place.     Next  boil  a  similar  cane  sugar  solution  with  a  few  drops 


26  PHYSIOLOGICAL    CHEMISTRY. 

of  dilute  hydrochloric  or  sulphuric  acid  several  minutes,  neutralize  with  sodium 
carbonate,  and  then  apply  the  Fehling  test.  The  characteristic  red  precipitate  now 
appears.  In  this  reaction  the  cane  sugar  is  broken  up  by  the  acid  into  a  molecule 
of  glucose,  and  a  molecule  of  fructose,  both  reducing  sugars  as  explained  above. 

The  behavior  of  cane  sugar  solutions  with  polarized  light  is  charac- 
teristic and  affords  the  simplest  and  most  accurate  means  for  quanti- 
tative determination.  The  specific  rotation  is  practically  independent 
of  the  concentration  and  is  represented  by  the  formula  [a]  D  =+66.5°. 

Strong  solutions  of  cane  sugar,  "  syrups,"  are  used  in  the  house- 
hold and  in  pharmacy  to  prevent  fermentation.  Hence  the  use  of  this 
sugar  in  the  canning  or  preserving  of  fruit. 

Lactose.  This  is  the  characteristic  sugar  of  all  kinds  of  milk, 
with  possibly  one  or  two  exceptions.  It  may  be  separated  from  the 
"whey"  which  is  the  product  remaining  after  skimming  and  precipi- 
tating the  casein.  It  is  made  commercially  in  large  quantities  as  a 
by-product  in  the  cheese  industry,  and  in  pure  crystallized  form  has 
the  formula  C^H^On-H^O. 

Milk  sugar  resembles  cane  sugar  in  respect  to  the  conditions  under 
which  it  may  be  fermented,  but  it  is  a  reducing  sugar  directly,  acting 
strongly  on  copper  or  bismuth  solutions.  In  its  behavior  with  polar- 
ized light  it  resembles  glucose  closely,  having  a  specific  rotation, 
[a]D=  -f-  52. 50.  Inverted  milk  sugar  ferments  readily,  and  products 
known  as  kumyss,  from  mare's  milk,  and  kephir,  from  cow's  milk, 
are  made  in  this  way.  In  digestion  lactose  splits  up  into  glucose  and 
galactose  readily,  while  cane  sugar  yields  glucose  and  fructose,  but 
less  readily.     With  phenyl  hydrazine  a  yellow  lactosazone  is  formed. 

Milk  sugar  is  much  less  soluble  in  water  than  is  cane  sugar  and  has 
but  a  slightly  sweet  taste.  It  is  used  mainly  in  the  production  of 
infant  and  invalid  foods  and  in  manufacturing  pharmacy  in  tablets, 
pills,  etc. 

Maltose,  or  the  sugar  of  malt,  is  produced  by  the  action  of  malt 
diastase  on  starch.  It  therefore  occurs  in  germinating  seeds  and 
grains,  and  is  present  wherever  a  diastase  acts  on  starch.  In  the 
action  of  weak  acids  on  starch  paste  malt  sugar  is  produced  as  a 
transition  stage,  glucose  finally  resulting  by  inversion.  In  this  country 
malt  sugar  is  not  a  common  article  of  commerce,  but  in  several  Euro- 
pean countries  it  has  been  produced  in  considerable  quantities  to  be 
used  as  an  article  of  food  in  the  place  of  glucose  or  cane  sugar.  The 
manufacture  of  a  relatively  pure  sugar  by  the  use  of  malt  diastase 
and  a  starchy  material,  such  as  corn,  seems  to  be  attended,  however, 
with  great  practical  difficulties. 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  27 

Maltose  is  readily  soluble  in  water,  sweet  but  not  to  the  same 
degree  as  cane  sugar,  and  is  not  directly  fermentable.  But  an  invert- 
ing enzyme  in  common  yeast  changes  it  so  quickly  that  it  was  long 
classed  among  the  true  fermenting  sugars.  The  view  is  now  gener- 
ally held  that  the  disaccharides  must  first  be  converted  into  mono- 
saccharides before  real  fermentation  can  take  place.  In  the  industries 
malt  sugar  is  thus  fermented  on  the  large  scale.  Toward  oxidizing 
solutions  its  behavior  is  like  that  of  glucose,  although  its  reducing 
power  is  not  quite  as  great.  With  phenyl  hydrazine  it  forms  a  malt- 
osazone,  and  on  polarized  light  its  rotating  power  is  very  great,  the 
specific  rotation  being  at  200  C.  [a]0  =  +  1370.  In  the  body  maltose 
is  changed  into  glucose  by  action  of  an  inverting  enzyme  occurring 
both  in  the  pancreas  and  in  the  true  intestinal  juice.  This  inversion 
seems  to  take  place  much  more  readily  than  in  the  case  of  cane  sugar, 
which  is  a  fact  of  considerable  physiological  importance. 

Isomaltose.  This  is  a  sugar  which  has  been  made  by  the  action  of 
fuming  hydrochloric  acid  on  glucose.  It  also  accompanies  the  true 
maltose  in  the  products  formed  by  the  action  of  diastase  on  starch. 
It  differs  from  maltose  in  rotating  power,  which  is  much  less,  in  the 
character  of  its  phenyl  osazone,  and  in  water  solubility.  It  reduces 
copper  and  bismuth  solutions  but  undergoes  fermentation  with  yeast 
very  slowly. 

Other  disaccharides  known  have  but  little  importance.  Mycose  or  trehalose  is 
found  in  certain  fungi,  agarose  is  obtained  from  the  juice  of  the  agave  plant. 
Melibiose  and  turanose  are  formed  in  the  hydrolysis  of  certain  polysaccharides. 

The  Saccharotrioses  or  Trisaccharides.  This  group  contains  a 
few  sugars  and  but  one  of  these  is  important  at  the  present  time. 

Melitose  or  raffinose.  This  sugar,  having  the  formula  C1SH32016 
-f-  5H20,  is  found  in  certain  kinds  of  manna  and  also  in  sugar  beets 
in  small  amount.  It  is  characterized  by  having  a  strong  rotation, 
la]D=  I04-5°-  Being  more  soluble  than  saccharose  it  is  found  in  the 
last  crystallizations  from  beet  juice,  and  thus  sometimes  contaminates 
the  beet  sugar.  Its  high  rotation  may  cause  an  error  in  the  estima- 
tion of  sugar  by  the  polarimeter.  When  inverted  with  acids  it  yields 
fructose,  and  the  disaccharide  melibiose. 

THE  DETERMINATION   OF   SUGARS. 
This  is  carried  out  in  several  ways.     In  one  method  the  reactions 
depend  on  the  reducing  power  of  sugars  on  alkaline  copper  or  other 
metallic  solutions.    The  Fehling  reagent  is  usually  employed.    Methods 
with  the  polariscope  will  be  described  later. 


28  PHYSIOLOGICAL    CHEMISTRY. 

Method  with  Fehling's  Solution.  Fehling's  solution,  as  described 
above,  is  made  arbitrarily  of  such  a  strength  that  one  cubic  centimeter 
is  reduced  by  5  milligrams  of  glucose,  on  the  supposition  that  the 
sugar  and  copper  salt  react  on  each  other  in  the  proportion  of  one 
molecule  of  glucose  to  five  molecules  of  crystallized  copper  sulphate. 

It  was  formerly  held  that  the  reaction  was  a  perfectly  definite  and 
simple  one,  and  could  be  expressed  in  this  manner,  but  it  is  now 
known  that  the  dilution  of  the  solutions  is  a  very  important  factor  in 
determining  the  amount  of  copper  reduced.  The  best  conditions  to  be 
employed  in  practice  have  been  determined  by  Soxhlet,  who  found  the 
reducing  power  of  several  sugars  to  vary  as  follows,  when  they  were 
tested  in  solutions  of  1  per  cent  strength : 

0.5  gm.  of  invert  sugar  in  1  per  cent  solution  reduces  101.2  cc.  of  Fehling's 
solution,  undiluted. 

0.5  gm.  of  invert  sugar  in  1  per  cent  solution  reduces  97.0  cc.  of  Fehling's 
solution,  diluted  with  4  volumes  of  water. 

0.5  gm.  of  glucose  in  1  per  cent  solution  reduces  105.2  cc.  of  Fehling's  solution, 
undiluted. 

0.5  gm.  of  glucose  in  1  per  cent  solution  reduces  101.1  cc.  of  Fehling's  solution, 
diluted  with  4  volumes  of  water. 

0.5  gm.  of  milk  sugar  in  1  per  cent  solution  reduces  74  cc.  of  Fehling's  solution, 
undiluted.     The  reducing  power  in  diluted  solution  is  the  same. 

0.5  gm.  of  maltose  in  1  per  cent  solution  reduces  64.2  cc.  of  Fehling's  solution, 
undiluted. 

0.5  gm.  of  maltose  in  1  per  cent  solution  reduces  67.5  cc.  of  Fehling's  solution, 
diluted  with  4  volumes  of  water. 

The  oxidizing  power  of  1  cc.  of  Fehling's  solution  with  each  kind  of  sugar  may 
be  tabulated  as  follows,  assuming  the  sugars  to  be  in  solutions  of  approximately 
1  per  cent  strength  when  acted  upon. 

One  cubic  centimeter  of  Fehling's  solution  oxidizes : 

When  When  Diluted  with 

Undiluted.  4  Vols,  of  Water. 

Glucose    4.75  mg.  4.94  mg. 

Invert  Sugar   4.94     "  5.15     " 

Milk   Sugar    6.76     "  6.76     " 

Maltose    7.78     "  7.40     " 

The  practical  application  of  the  test  is  best  shown  by  an  experiment. 

Experiment.  Measure  out  accurately  into  a  flask  holding  about  250  cc,  25  cc. 
of  the  copper  solution  and  the  same  volume  of  the  alkaline  tartrate.  Heat  the 
mixture,  or  Fehling's  solution,  on  a  wire  gauze  and  note  that  it  remains  clear.  Fill 
a  50  cc.  burette  with  a  dilute  glucose  solution  and  run  10  cc.  into  the  hot  liquid. 
Boil  one  minute,  shaking  the  flask  continuously,  and  allow  the  mixture  to  settle.  If 
the  supernatant  liquid  appears  yellow  this  indicates  that  the  sugar  solution  is  much 
too  strong  and  must  be  diluted  with  at  least  an  equal  volume  of  water  before  begin- 
ning another  test.  If,  on  the  other  hand,  the  liquid  is  still  blue,  add  2  cc.  more  of 
the  sugar  solution,  boil  again  for  a  minute  and  allow  to  settle.  If  the  color  is  now 
yellow  an  approximate  value  for  the  amount  of  sugar  in  the  solution  becomes 
known,  but  if  still  blue,  the  operation  of  adding  solution  and  boiling  must  be  con- 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  29 

tinued  until,  after  settling,  a  yellow  color  appears.  Approximately  250  mg.  of 
glucose  is  required  to  reduce  the  Fehling  solution  taken,  and  this  must  be  con- 
tained in  the  sugar  solution  added.  From  this  preliminary  experiment  calculate  the 
amount  of  sugar  present  in  each  cubic  centimeter. 

Experiment.  With  the  data  obtained  in  the  above  experiment  as  a  basis,  make 
now  a  new  sugar  solution,  having  a  strength  of  about  1  per  cent.  Measure  out 
50  cc.  of  the  Fehling's  solution,  heat  to  boiling  and  run  the  new  sugar  solution  from 
the  burette  as  before,  the  first  addition  being  about  20  cc.  Boil  and  note  the  color 
after  settling  and  then  cautiously  continue  the  addition  of  sugar  solution,  a  few 
tenths  of  a  cc.  at  a  time,  boiling  after  each  addition,  until  the  blue  color  gives  place 
to  a  yellowish  green  and  then,  by  the  addition  of  a  drop  or  two,  to  a  pale  yellow. 

Sometimes  the  final  disappearance  of  the  copper  from  the  solution  is  determined 
by  filtering  a  few  drops  through  a  very  small  filter  and  adding  a  drop  of  acetic 
acid  and  a  drop  of  ferrocyanide  solution  to  the  filtrate,  when  the  characteristic 
reddish  color  is  given  if  a  trace  of  copper  is  present. 

To  determine  cane  sugar  by  the  Fehling's  solution  it  must  first  be  converted  or 
"  inverted "  into  a  mixture  of  glucose  and  fructose.  If  the  sugar  is  in  the  dry 
condition  the  inversion  can  be  accomplished  as  follows :  Weigh  out  9.5  gm.,  dis- 
solve in  700  cc.  of  water,  add  20  cc.  of  normal  hydrochloric  acid  and  heat  for  30 
minutes  on  the  water-bath.  Then  neutralize  with  20  cc.  of  normal  sodium  hydroxide 
solution  and  make  up  to  1000  cc.  on  cooling.  This  gives  now  a  1  per  cent  solution, 
which  is  employed  as  given  for  glucose,  using  the  factor  4.94  instead  of  4.74  as 
the  amount  of  sugar  oxidized  by  each  cc.  of  the  copper  solution.  On  completion 
of  the  experiment  calculate  95  parts  of  cane  sugar  for  each  100  parts  of  invert 
sugar  found. 

The  attention  of  the  student  is  directed  to  the  fact  that  malt  sugar  and  milk 
sugar  may  be  determined  by  the  aid  of  Fehling's  solution  without  previous  inver- 
sion.    This  should  be  verified  by  experiment. 

Method  by  Use  of  Ammoniacal  Copper  Solutions.  The  deter- 
mination of  glucose  in  pure  aqueous  solution  by  the  above  method  is 
simple  and  accurate,  but  in  liquids  containing  other  organic  matters 
the  precipitate  sometimes  fails  to  settle  readily,  so  that  the  recognition 
of  the  end  point  is  difficult.  This  is  often  the  case  with  urine  and 
other  physiologically  important  liquids.  Advantage  may  be  taken  of 
the  fact  that  cuprous  oxide  dissolves  in  ammonia  without  color  to  pre- 
pare a  quantitative  solution  with  which  this  difficulty  may  be  largely 
overcome.  Pavy  was  the  first  to  employ  such  a  reagent  practically 
and  his  solution  was  made  by  diluting  the  ordinary  Fehling's  solu- 
tion with  ammonia  in  certain  proportions.  His  suggestion  has  re- 
ceived several  modifications.  In  all  of  these  the  weak  sugar  solution 
is  added  to  the  boiling  ammoniacal  copper  solution  until  the  color  of 
the  latter  is  just  discharged,  at  which  point  the  reduction  of  the  copper 
hydroxide  by  the  sugar  is  complete.  In  place  of  using'  the  Fehling's 
solution  it  is  well  to  make  the  Loewe  solution  with  glycerol  the  basis 
of  the  dilution.  A  solution  of  this  kind  may  be  made  by  the  formula 
below,  in  which  the  proportions  have  been  found  by  the  present  writer 
to  give  the  best  result  in  practical  work.  One  cubic  centimeter  of  the 
solution  oxidize-,  one  milligram  of  glucose  in  0.2  per  cent  solution. 


30 


PHYSIOLOGICAL    CHEMISTRY. 


It  is  made  with  the  following  amounts  per  liter : 

Copper  sulphate,  cryst    8.166  gm. 

Sodium  hydroxide  (100  per  cent)    15.000 

Glycerol    25.000  cc. 

Ammonia  water,  0.9  sp.  gr  350.000 

Water  to  make   1,000.000     " 

Experiment.  Of  this  solution,  measure  50  cc.  into  a  flask  and  dilute  with  water 
to  100  cc.  To  prevent  too  rapid  an  escape  of  ammonia  and  avoid  reoxidation  to 
some  extent,  add  to  the  mixture,  while  warming,  enough  pure  white  solid  paraffin 
to  make  a  layer  3  or  4  millimeters  in  thickness  when  melted.  The  burette  tip  for 
discharging  the  sugar  solution  is  made  long  enough  to  pass  down  the  neck  of  the 
flask  and  below  this  paraffin.  By  boiling  gently  and  adding  the  weak  saccharine 
liquid  slowly,  very  close  and  constant  results  may  be  obtained.  At  the  end  of  the 
titration  the  paraffin  is  solidified  by  inclining  the  flask  and  immersing  it  in  cold 
water,  or  by  flowing  cold  water  over  it.  The  reduced  liquid  is  then  poured  out 
and  the  cake  of  paraffin  is  thoroughly  washed  for  the  next  test.  A  flask  so  pre- 
pared may  be  used  for  a  hundred  titrations.  To  prevent  bumping  and  facilitate 
easy  and  uniform  boiling,  it  is  well  to  add  a  few  very  small  fragments  of  pumice- 
stone. 

A  solution  made  as  above  is  not  too  strong  in  copper  for  accurate  work,  but  the 
volume  of  ammonia  necessary  to  hold  a  much  larger  amount  of  the  reduced  oxide 
in  solution  would  render  the  process  very  inconvenient.  Some  practice  is  neces- 
sary to  show  just  how  fast  the  saccharine  solution  may  be  safely  added.  If  added 
too  rapidly  the  end  point  may  be  overlooked  and  the  sugar  content  made  to 
appear  too  low. 

Polarization  Tests  and  the  Use  of  the  Polariscope.  This  is  the  proper  place 
to  show  the  applications  of  the  polariscope  in  the  examination  of  sugars  and  other 
substances.  For  a  description  of  the  various  forms  of  polariscopes  and  discussion 
of  the  optical  principles  involved  in  their  construction  the  reader  is  referred  to  the 


Fig.  1.    A  common  form  of  Laurent  polariscope.     The  polarizing  prism  is  situated  in  the 
tube  below  H,  the  analyzer  at  E,  B  is  the  reading  microscope  and  C  a  vernier. 


author's  translation  of  Landolt's  work,  "The  Optical  Rotation  of  Organic  Sub- 
stances and  its  Practical  Applications,"  but  a  few  words  of  elementary  explanation 
may  be  in  order  here.  A  simple  form  of  polarimeter  in  common  use  is  shown  in 
the  illustrations. 

In  the  polariscopes  in  common  use  for  general   scientific   studies  homogeneous 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  3  I 

yellow  light  is  employed  and  this  is  first  polarized  by  passing  through  a  specially 
designed  prism  in  the  front  part  of  the  instrument.  This  prism  is  usually  some 
form  of  a  Nicol  prism  and  is  so  constructed  that  only  one  of  the  polarized  rays 
produced  at  the  start  is  allowed  to  emerge  and  pass  through  the  instrument.  The 
plane  in  which  this  ray  vibrates  is  called  the  plane  of  polarization.  Such  plane 
polarized  light  passes  through  air,  water,  alcohol,  ether,  glass  and  many  other  trans- 
parent substances  without  change ;  that  is  the  direction  in  which  the  light  vibrates 
remains  unaltered.  But  many  organic  substances,  liquids  or  solids  dissolved,  have 
the  remarkable  property  of  causing  this  plane  of  polarization  to  change  direction; 
in  other  words  the  plane  of  vibration  of  the  light  suffers  a  twist  or  rotation  in 
passing  through  a  column  of  the  liquids.  Substances  which  have  the  power  of 
changing  the  direction  of  the  plane  of  vibration  of  polarized  light  passing  through 
them  are  called  "  active  "  substances  and  the  extent  of  the  rotation  is  dependent  on 


GO 


■* ^ 

Fig.  2.  This  represents  the  course  of  the  light  through  the  Laurent  polariscope,  the 
direction  being  reversed,  however,  from  that  of  the  last  figure.  a  is  a  bichromate  plate 
to  purify  the  light,  b  the  polarizing  Nicol,  c  a  thin  quartz  plate  covering  half  the  field 
and  essential  in  producing  a  second  polarized  plane,  d  the  tube  to  contain  the  liquid 
under  examination,  e  the  analyzing  Nicol  and  /  and  g  the  ocular  lenses. 

the  number  of  molecules  which  the  light  passes.  In  the  case  of  homogeneous 
liquids  like  oil  of  turpentine  the  rotation  varies  with  the  length  of  the  column 
through  which  the  light  must  pass,  while  in  the  case  of  dissolved  solids,  sugar 
solutions  for  example,  the  amount  of  the  twist  or  rotation  varies  with  the  length 
of  the  column  of  solution,  and  also  with  its  concentration  or  number  of  molecules 
in  a  given  volume.  An  instrument  which  has  some  device  which  enables  the 
observer  to  read  off  this  rotation  in  degrees  is  called  a  polarimeter,  and  the  number 
of  degrees  read  constitutes  a  measure  of  the  strength  or  concentration  of  the 
substance. 

In  order  to  compare  the  rotation  of  substances  the  term  "  specific  rotation " 
has  been  introduced.  This,  as  applied  to  liquids,  may  be  defined  as  the  rotation 
which  a  substance  would  exhibit  if  examined  in  a  column  ioo  millimeters  in  length 
having  a  concentration  of  i  gram  of  active  substance  to  each  cubic  centimeter. 
This  rotation  must  therefore  be  a  calculated  one,  and  is  found  as  illustrated  by  this 
concrete  case.  Consider  a  solution  made  by  dissolving  25  gm.  of  pure  cane  sugar 
in  distilled  water  and  diluted  to  make  exactly  100  cc.  This  is  then  examined  in  a 
polarization  tube,  which  is  a  long  tube  of  glass  or  metal  having  ends  of  plane 
polished  glass  perfectly  parallel  to  each  other.  The  sugar  solution  forms  then  a 
clear  transparent  column  of  definite  length,  which,  assume  in  this  case,  is  200 
millimeters.  By  examination  in  the  polarimeter  it  is  found  now  that  this  solution 
rotates  the  plane  of  polarized  sodium  light  through  33. 250.  For  a  solution  with  100 
grams  to  100  cc.  the  rotation  by  calculation  should  be  four  times  this,  or  1330,  in  the 
200  mm.  tube  or  66.50  in  the  100  mm.  or  standard  tube.  This  is  then  the  specific 
rotation,  and  we  express  it  by  the  formula: 

[a]  D  =  66.5°, 

in  which  [a]  is  the  usual  symbol  for  the  specific  rotation,  and  the  D  the  indication 


32  PHYSIOLOGICAL    CHEMISTRY. 

that  the  observation  is  made  with  sodium  light,  a  without  the  brackets  is  the 
angle  of  rotation  as  read  off.  Remembering  the  definition  of  specific  rotation  we 
have  this  general  formula  as  applied  to  solutions : 

,       ioo  X  iooa      io4a 

[a]=    ixc    -rr 

in  which  /  expresses  the  length  of  the  observation  tube  in  millimeters  and  c  the 
concentration  or  strength  of  the  solution  in  grams  per  ioo  cubic  centimeters. 

For  many  substances  this  rotation  is  so  characteristic  and  so  easily  observed  that 
it  constitutes  a  good  test  of  purity  or  identity.  With  the  specific  rotation  known 
the  following  relation  enables  us  to  find  the  amount  of  active  substance  in  solution : 

io4q 

C~[a]-l 

The  following  are  some  specific  rotations  which  have  importance  from  the  stand- 
point of  physiological  chemistry,  the  temperature  being  200  C.  in  each  case : 

Cane  sugar,  [a]x)  =  -|-   66.50  c  =  10  to  30 

Milk  sugar  (+H20),  [a]D  =  +   52.5°  c=   3  to  40 

Malt  sugar   (+H,0),  [a]D  =  +  137.0°  c—   2  to  20 

Glucose,  [oi]d  =  -\-   53-0°  c  =  20 

Levulose,  [a]o  =  —   93.00  c=  10  to  20 

Invert  sugar,  [a]x>  =  — ■  20.20  c  =  15 

The  protein  substances,  dextrin,  glycogen  and  a  number  of  other  compounds  to  be 
referred  to  later  have  also  a  high  rotating  power,  which  finds  application  in 
investigations. 

THE  POLYSACCHARIDES. 

We  have  here  a  very  important  group  of  bodies,  some  of  which 
appear  to  have  an  extremely  complex  structure.  Formerly  these  com- 
pounds were  assumed  to  be  simpler  than  the  sugars  and  were  repre- 
sented by  the  general  formula  C6H10O5.  The  action  of  water  in 
producing  glucose  was  assumed  to  consist  merely  in  the  addition  of 
one  molecule  as  shown  by  the  formula : 

CJJttO,  +  H,0  =  CiHfflO» 

But  this  view  is  no  longer  held;  the  starches,  cellulose  bodies  and 
certain  gums  belonging  to  the  group  have  been  shown  to  exist  in  the 
form  of  large  and  probably  very  complex  molecular  aggregations,  and 
the  formula  (C6H10O5)n  is  now  usually  employed  to  indicate  this  fact. 

These  polysaccharides  are  related  to  the  real  sugars  by  several 
reactions.  By  certain  treatment  most  of  them  may  be  converted  more 
or  less  readily  into  maltose,  glucose  or  fructose,  and  besides  this  they 
yield  the  ester  derivatives  characteristic  of  polyhydric  alcohols.  In 
their  natural  condition  they  are  mostly  insoluble  in  water  and  other 
solvents.  It  is  customary  to  make  three  classes  of  these  compounds,  of 
which  the  starches  or  amyloses,  as  the  most  important,  will  be  treated 
first. 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  33 

The  Amyloses.  In  the  vegetable  kingdom  starch  is  a  common 
and  widely  distributed  reserve  material,  a  sugar,  probably  saccharose, 
being  first  formed  as  a  synthetic  product.  Starch  is  found  in  many 
seeds,  grains  and  tubers  in  the  form  of  minute  granules  which  are 
often  characteristic  in  shape  or  size.  They  may  be  extremely  small, 
pepper  starch  for  example,  or  relatively  large,  as  in  the  case  of  arrow- 
root starch.  Under  the  microscope  these  granules  often  appear  to  be 
built  up  of  concentric  layers,  and   furthermore  they  are  not  homo- 


Fig.  3.     Wheat  starch  magnified  about  350  diameters. 

geneous  in  composition.  The  outer  part  of  the  granule  consists  of  a 
covering  or  sheath  of  starch  cellulose  within  which  is  the  large  mass  of 
starch  granulose.  The  cellulose  sheath  is  insoluble  in  water  at  the 
ordinary  temperature,  but  with  elevation  of  temperature  in  presence 
of  an  excess  of  water  the  protective  layer  breaks  and  allows  the 
granulose  to  form  a  more  or  less  perfect  solution  of  so-called  soluble 
starch. 

On  the  technical  scale  starch  may  be  obtained  from  a  variety  of  sub- 
stances. The  common  sources  are  potatoes,  corn,  rice  and  arrowroot. 
The  manufacture  is  largely  a  mechanical  operation,  which  may  be 
illustrated  as  follows : 

Experiment.  Grate  a  potato  to  a  pulp  by  means  of  an  ordinary  tin  grater,  mix 
the  pulp  with  water  and  squeeze  through  a  piece  of  coarse  unbleached  muslin, 
collecting  the  strained  liquids  in  a  large  beaker.  Allow  the  mixture  to  settle  a 
half  hour  or  longer  and  pour  off  the  water,  which  contains  some  soluble  albuminous 

4 


34  PHYSIOLOGICAL    CHEMISTRY. 

substances,  some  cellular  floating  matter,  but  very  little  starch.  Most  of  this  will 
be  found  in  the  bottom  of  the  beaker.  Add  some  fresh  water,  stir  up  and  allow 
to  settle.  Now  pour  the  water  off  again  and  repeat  these  operations  until  the 
starch  appears  perfectly  clean  and  white.  Transfer  this  starch  to  a  clean  shallow 
dish  and  allow  what  is  not  intended  for  immediate  use  to  dry  spontaneously  in  an 
atmosphere  free  from  dust.  The  dried  product  will  consist  of  minute  glistening 
particles  resembling  small  crystals.     Save  this  starch  for  tests  given  below. 

Experiment.  Examine  starch  from  several  sources  under  the  microscope,  em- 
ploying a  power  of  about  300  diameters.  Clean  a  glass  slide  thoroughly,  place  in 
the  center  of  it  a  small  drop  of  water,  and  stir  into  this  by  means  of  a  needle,  or 
glass  rod,  a  minute  quantity  of  starch.  Now  drop  on  a  clean  cover  glass  in  such 
a  manner  as  to  exclude  air  bubbles,  and  place  under  the  microscope  for  observation. 

Experiment.  Repeat  the  last  experiment,  using  an  aqueous  solution  of  iodine 
instead  of  water.  The  starch  granules  will  now  appear  blue.  For  the  detection 
of  starch  in  mixtures  the  use  of  iodine  is  often  indispensable. 

Some  idea  of  the  size  of  the  starch  cells  may  be  obtained  from  this 
table  which  gives  the  mean  diameter  in  fractions  of  a  millimeter. 
For  starch  granules  which  are  oval  instead  of  circular,  the  averages 
of  the  longer  and  shorter  diameters  is  given: 

Potato 0.06  -0.10 

Common  arrowroot  0.01  -0.07 

Corn   0.007-0.02 

Wheat 0.002-0.05 

Rice  0.005-0.008 

Pea   0.016-0.028 

Bean    0.035 

Barley  0.013-0.040 

Rye   0.002-0.038 

Starch  may  be  recognized  by  a  number  of  chemical  tests,  the  best  of  which  are  the 
following : 

Experiment.  Boil  a  small  amount  of  starch  with  water  so  as  to  make  a  thin 
paste.  Allow  this  to  cool,  and  add  a  few  drops  of  an  aqueous,  or  alcoholic  solu- 
tion, of  iodine.  A  deep  blue  color  is  formed,  which  disappears  on  boiling  the 
mixture.  This  test  is  exceedingly  delicate  and  characteristic,  and  serves  for  the 
detection  of  minute  traces  of  iodine  as  well  as  starch.  The  blue  color  is  destroyed 
by  alkalies  or  much  alcohol  as  well  as  by  heat. 

Experiment.  That  starch  is  insoluble  in  cold  water  may  be  shown  by  stirring 
some  with  water  in  a  beaker,  allowing  to  settle,  and  pouring  the  liquid  through  a 
paper  filter.  The  filtrate  tested  with  the  iodine  solution  does  not  give  a  blue  color. 
Use  the  potato  starch  of  the  experiment  for  this  test. 

When  boiled  with  dilute  acids  starch  is  converted  into  soluble  com- 
pounds. The  nature  of  these  compounds  depends  on  the  acid  used 
and  on  the  duration  of  the  heating.  By  prolonged  heating  glucose  is 
the  main  product  of  the  reaction,  as  already  illustrated,  but  various 
intermediate  steps  may  be  recognized,  maltose  and  forms  of  dextrin 
being  readily  demonstrated.  With  strong  acids  the  results  are  quite 
different.  With  sulphuric  acid  the  reaction  is  completely  destructive, 
water,    carbon   dioxide   and   sulphurous   acid    from   reduction   being 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  35 

formed.  Strong  nitric  acid  acts  as  an  oxidizing  agent  and  by  proper 
manipulation  oxalic  acid  may  be  obtained  in  quantity  as  a  product  of 
the  oxidation. 

Experiment.  Add  15  cc.  of  strong  sulphuric  acid  to  a  gram  of  starch  in  a  flask 
holding  about  200  cc.  Heat  to  the  boiling  point  and  observe  that  a  black  mass  is 
soon  produced.  By  prolonged  heating  this  is  further  decomposed,  while  fumes  of 
sulphurous  oxide  escape,  leaving  finally  a  colorless  liquid. 

Experiment.  Add  15  cc.  of  strong  nitric  acid  to  one  gram  of  starch  in  a  flask 
holding  200  to  300  cc,  place  this  on  a  sand-bath  in  a  fume  chamber  and  apply 
heat.  After  a  time  copious  red  fumes  are  given  off.  Remove  the  lamp  and  allow 
the  reaction  to  continue  until  the  fumes  cease  to  be  evolved.  Finally,  transfer  the 
liquid  to  a  porcelain  dish  and  evaporate  to  a  small  volume.  On  cooling,  a  crystalline 
residue  remains  which  consists  mainly  of  oxalic  acid. 

When  carefully  heated  starch  may  be  largely  converted  into  a  form 
of  dextrin,  which,  as  will  be  fully  explained  later,  is  one  of  the  impor- 
tant stages  in  the  common  transformations  of  starch.  The  reaction 
is  employed  on  the  large  scale  in  the  manufacture  of  British  gum 
which  is  used  in  the  preparation  of  size  and  paste  for  various  purposes. 

Experiment.  Heat  about  10  gm.  of  starch  in  a  porcelain  dish  on  a  sand-bath  to 
a  temperature  short  of  the  point  where  it  begins  to  scorch.  It  is  necessary  to  stir 
well  all  the  time,  and  continue  the  heat  ten  minutes  after  the  starch  has  become 
uniformly  yellowish  brown.  Then  allow  the  dish  to  cool,  add  water  and  boil 
thoroughly,  which  brings  part  of  the  product  into  solution.  When  sufficiently 
diluted  this  solution  can  be  filtered.  The  filtrate  is  precipitated  by  alcohol.  The 
addition  of  a  few  drops  of  iodine  solution  to  the  aqueous  liquid  gives  rise  to  a 
reddish  color  characteristic  of  dextrin. 

The  chief  uses  of  starch  have  been  referred  to  in  other  connections. 
Much  is  directly  employed  as  food  and  large  quantities  are  converted 
into  glucose  as  shown  above.  The  production  of  various  kinds  of 
dextrin  and  British  gum  is  also  extremely  important  and  consumes 
enormous  quantities  of  starch.  In  the  form  in  which  it  occurs  in 
nature,  that  is  mixed  with  other  substances  in  small  amount,  starch  is 
the  most  abundant  of  our  foodstuffs,  and  the  one  consumed  in  largest 
amount.  Great  interest  therefore  attaches  to  the  reactions  by  which 
this  starch  is  made  soluble  or  digested  as  a  step  in  its  assimilation. 
The  discussion  of  this  fundamental  point  will  be  left,  however,  for  a 
following  chapter,  when  the  theory  of  digestive  operations  can  be 
explained  as  a  whole. 

In  certain  plants  a  variety  of  starch  called  inulin  occurs.  It  is  best 
obtained  from  tubers  of  the  dahlia,  and  is  interesting  from  the  fact 
that  by  hydrolysis  it  yields  fructose  instead  of  glucose.  It  differs  from 
the  ordinary  starch  in  yielding  a  true  solution  with  hot  water,  and  in 
giving  a  yellow  instead  of  a  blue  color  with  iodine. 


36  PHYSIOLOGICAL    CHEMISTRY. 

Glycogen,  or  animal  starch.  This  product,  which  is  formed  in 
the  liver,  is  related  in  many  ways,  both  chemically  and  physiologically, 
to  common  starch.  In  some  respects  it  resembles  also  the  simple 
sugars,  from  which  it  is  indeed  derived,  and  may  be  said  to  stand 
between  them  and  vegetable  starch.  It  is  readily  soluble  in  water, 
giving,  however,  an  opalescent  solution.  This  is  especially  character- 
ized by  a  strong  action  on  polarized  light,  [a]D  =  +  1960  to  2130, 
according  to  different  authorities.  Like  common  starch  glycogen  is 
a  reserve  material,  being  formed  from  the  absorbed  sugar  of  the 
digestive  process,  and,  in  turn,  being  reconverted  into  sugar  from  the 
liver  as  this  is  required  for  oxidation  in  the  body.  After  death  the 
store  of  glycogen  in  the  liver  rapidly  diminishes,  glucose  being  pro- 
duced. The  amount  of  glycogen  present  in  the  liver  varies  greatly 
with  the  diet  and  time  after  eating.  It  may  make  up  12  to  16  per 
cent  of  the  total  weight  of  the  organ,  but  is  usually  much  below  this, 
perhaps  in  the  mean  2  to  3  per  cent.  In  addition  to  its  occurrence  in 
the  liver  glycogen  is  found  in  variable  amount  in  the  muscles,  and  in 
traces  in  other  body  tissues.  It  occurs  also  in  the  vegetable  kingdom, 
and  has  been  recognized  in  certain  fungi.  The  laboratory  production 
of  glycogen  and  some  of  the  simple  reactions  are  illustrated  by  the 
following  experiments,  while  the  physiological  relations  will  be 
reserved  for  further  discussion  in  a  later  chapter. 

Experiment.  Kill  a  rat  or  a  rabbit  which  has  been  well  fed;  remove  the  liver 
as  quickly  as  possible,  and  without  delay  cut  it  into  small  bits,  which  throw  into  a 
vessel  of  boiling  water.  The  weight  of  the  water  should  be  about  five  times  that 
of  the  minced  liver.  Boil  five  minutes,  then  remove  from  the  water  and  rub  up 
in  a  mortar  with  fine  clean  quartz  sand.  In  this  way  the  fragments  of  liver  be- 
come thoroughly  disintegrated.  The  contents  of  the  mortar,  sand  as  well  as  liver, 
are  thrown  into  boiling  water  again  and  kept  at  ioo°  15  minutes.  At  the  end  of  this 
time  enough  dilute  acetic  acid  must  be  added  to  impart  a  faint  acid  reaction.  This 
coagulates  and  precipitates  some  albuminous  matters,  which  are  separated  when  the 
hot  mixture  is  filtered.  In  the  opalescent  filtrate,  which  must  be  collected  in  a 
cold  beaker,  a  further  precipitation  of  albuminous  matter  is  effected  by  adding  a 
few  drops  of  hydrochloric  acid  and  some  potassium  mercuric  iodide  as  long  as  a 
precipitate  forms. 

Filter  again  and  use  the  dilute  aqueous  solution  of  glycogen  resulting  for  tests 
below. 

Experiment.  Evaporate  about  half  of  the  liquid  above  to  a  small  volume  and 
precipitate  impure  glycogen  as  an  amorphous  white  powder  by  addition  of  strong 
alcohol. 

Experiment.  Add  a  little  tincture  of  iodine  to  a  small  portion  of  the  solution, 
and  note  the  red  color  produced.  This  color  is  discharged  by  heat.  Boil  some  of 
the  solution  with  dilute  hydrochloric  acid  ten  minutes;  neutralize  the  acid  nearly, 
-cool  and  again  test  with  iodine.  No  color  is  now  produced,  as  glycogen  has  dis- 
appeared under  the  treatment,  having  been  converted  into  sugar. 

It  has  been  remarked  above  that  after  death  the  store  of  glycogen  in  the  liver 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  Z7 

rapidly  disappears,  so  that  tests  applied  at  the  end  of  a  day  or  two  fail  to  show 
its  presence.     This  may  be  shown  as  follows : 

Experiment.  Cut  some  beef  liver  from  the  market  into  small  bits  and  extract 
with  boiling  water.  Boil  longer  to  coagulate  protein  bodies,  after  adding  some 
sodium  sulphate.  Apply  the  iodine  test  for  glycogen,  which  is  found  absent,  and 
the  Fehling  test  for  sugar,  which  is  found  present  in  quantity.  It  is  an  excellent 
exercise  also  to  determine  the  amount  of  sugar  which  may  be  obtained  from  a 
given  weight  of  liver.  In  this  test  the  extraction  must  be  repeated  with  several 
portions  of  water. 

The  Gums.  Some  of  these  occur  in  nature  as  products  related  to 
the  pentose  group  of  sugars  referred  to  some  pages  back.  Others  are 
related  to  starch,  and  on  transformation  yield  finally  hexoses.  The 
group  of  gums  includes  further  the  dextrin  bodies  formed  from  starch 
by  several  reactions,  one  of  which  has  been  already  illustrated.  The 
transformation  of  starch  by  the  action  of  weak  acids  or  enzymes  is 
far  from  being  a  simple  process  and  much  uncertainty  still  exists  as 
to  the  number  of  intermediate  products  between  the  parent  substance 
and  the  final  maltose  or  glucose.  Some  writers  have  attempted  to 
distinguish  several  well  defined  stages  in  the  reaction  and  describe  as 
definite  bodies  erythrodextrin,  achroodextrin,  amylodextrin  and 
nialtodcxtrin.  The  first  gives  a  violet-red  color  with  iodine  and  is 
easily  precipitated  by  alcohol ;  the  second  gives  no  color  with  iodine, 
but  is  still  precipitated  by  alcohol.  It  shows  reducing  power  with 
Fehling's  solution,  and  may  be  looked  upon  as  one  of  the  end  products 
of  the  action  of  diastase  on  starch,  maltose  being  the  other.  The 
name  amylodextrin  is  given  to  a  product  of  diastatic  action  and  also 
to  a  dextrin  formed  by  the  treatment  of  starch  with  very  dilute  acids. 
It  is  said  to  show  a  purple  color  with  iodine,  and  to  exhibit  very  strong 
dextro  rotation.  The  existence  of  maltodextrin  is  affirmed  by  several 
writers,  but  the  properties  of  the  substance  are  not  well  established. 
Some  authors  have  gone  so  far  as  to  recognize  several  modifications 
of  achroodextrin,  which  are  described  as  a,  /?  and  y  forms,  and  which 
differ  in  optical  properties  and  reducing  power.  The  more  recent 
extended  investigations  seem  to  disprove  this  notion,  however,  and  the 
most  that  can  be  safely  said  is  that  along  with  maltose  an  end  product 
is  produced  by  diastatic  conversion  of  starch  which  is  probably  a  single 
substance.  What  is  called  erythrodextrin  is  more  likely  to  be  a  mix- 
ture, possibly,  of  soluble  starch  and  achroodextrin.  Under  the  name 
crythrogramdose  a  similar  complex  has  been  described.  In  a  later 
chapter  on  the  action  of  ferments  more  will  be  said  about  the  theory 
of  the  transformation  of  starch  into  these  products. 

The  true  dextrins  are  not  directly  fermentable  with  yeast,  but  they 


38  PHYSIOLOGICAL    CHEMISTRY. 

appear  to  be  aldehyde  bodies  and  as  such  have  reducing  power.  They 
react  also  with  phenyl  hydrazine  and  yield  osazones,  which,  however, 
are  not  easily  purified,  because  of  their  solubility.  The  dextrins  have 
a  slightly  sweetish  taste  and  all  show  a  specific  rotation  about 
[a]D  =  -f-  1960.  Beyond  the  empirical  formula  C6H10O5  it  is  not 
possible  to  go  in  describing  the  constitution  of  these  bodies. 

The  natural  vegetable  gums  are  often  mixtures  of  several  sub- 
stances, and  but  few  of  them  have  been  studied.  Gum  arabic  and  gum 
Senegal  are  the  potassium  and  calcium  salts  of  arabic  acid  to  which 
the  formula  (C6H10O5)2  +  H20  is  given.  On  treatment  with  weak 
sulphuric  acid  both  arabinose  and  galactose  appear  to  be  formed. 
Agar-agar  is  said  to  yield  lactose  and  then  galactose,  while  cherry  gum 
yields  arabinose. 

Cellulose.  The  cell  walls  of  vegetable  substances  consist  of  cellu- 
lose mixed  always  with  related  compounds  of  which  a  body  called 
lignin  is  the  most  important.  The  cellulose  resists  the  action  of  strong 
oxidizing  or  other  agents  much  more  perfectly  than  do'  the  accom- 
panying bodies,  and  may  therefore  be  freed  from  them  by  various 
treatments.  In  washed  Swedish  filter  paper  we  have  an  illustration  of 
nearly  pure  cellulose,  as  all  the  other  bodies  in  the  original  fibers  have 
been  removed  by  the  bleaching  and  washing  processes  to  which  the 
raw  material  was  subjected  in  the  manufacture  of  the  paper.  A  pure 
cellulose  paper  may  be  made  from  wood  also,  but  only  by  more  com- 
plicated operations. 

The  pure  cellulose  is  characterized  by  insolubility  in  water,  weak 
acids,  alkalies,  alcohol  or  ether.  It  may  be  dissolved  rather  readily 
in  a  solution  known  as  Schweitzer's  reagent  and  by  prolonged  di- 
gestion with  acids  is  converted  into  hexoses  and  pentoses. 

The  natural  celluloses  may  be  divided  roughly  into  three  groups: 
(a)  those  which  resist  hydrolytic  action  very  perfectly  and  are  not 
capable  of  serving  as  foodstuffs  for  any  animals;  in  this  group  we 
have  linen  and  cotton  fibers,  hemp,  China  grass,  etc.  (b)  Those 
which  are  less  resistant  to  hydrolytic  action  and  which  contain  active 
CO  groups.  These  bodies  may  be  called  oxycelluloses ;  they  yield  also 
furfuraldehyde  by  distillation  with  hydrochloric  acid.  In  this  group 
we  have  the  mass  of  the  material  found  in  the  fundamental  tissues  of 
flowering  plants,  and  a  large  part  of  ordinary  woody  tissue.  This 
lignified  tissue  is  made  up  of  compound  celluloses  or  lignocelluloses 
from  which  the  cellulose  proper  may  be  isolated  in  a  variety  of  ways. 
Some  of  the  bodies  in  this  sroup  are  partly  digestible  and  have  some 
value  as  foods  for  the  herbivora.     (c)  In  this  third  group  we  have 


THE    CARBOHYDRATES    AND    RELATED    BODIES.  39 

substances  described  as  pseudocelluloses  or  hemicelluloses  and  which 
offer  little  resistance  to  hydrolysis.  They  are  easily  attacked  by  weak 
acids  and  alkalies  and  also  suffer  digestion  by  enzymes,  so  that  they 
may  be  classed  among  the  foodstuffs  of  limited  value  for  the  herbivora. 
These  bodies  resemble  starch  much  more  than  they  resemble  the  ligno- 
celluloses.  By  action  of  weak  acids  fermentable  sugars  are  formed 
almost  quantitatively  from  pseudocelluloses,  while  from  the  ligno- 
celluloses  not  over  about  20  per  cent  of  fermentable  sugars  may  be 
obtained. 

Experiment.  Prepare  Schweitzer's  reagent  by  first  precipitating  copper  hydrox- 
ide from  copper  sulphate  solution,  in  the  presence  of  a  little  ammonium  chloride, 
by  addition  of  sodium  hydroxide  in  excess.  Wash  the  precipitate  thoroughly  and 
then  dissolve  it  in  the  smallest  possible  quantity  of  strong  ammonia  water.  This 
yields  a  deep  blue  solution,  the  reagent  in  question.  It  dissolves  cotton  rather 
easily.  This  solution  may  be  filtered  after  dilution,  and  from  the  filtrate  a  pure 
cellulose  is  thrown  down  by  addition  of  acids. 

By  action  of  strong  nitric  acid,  aided  by  sulphuric  acid,  cellulose  is  converted  into 
a  series  of  nitrates  or  "  nitro-celluloses."  The  number  of  N03  groups  added  de- 
pends on  the  strength  of  the  acid  mixture  and  time  of  its  action.  The  more 
highly  nitrated  products  constitute  the  well-known  explosives.  Products  not  so 
highly  nitrated  are  used  in  the  preparation  of  collodion  and  celluloid.  This  latter 
is  essentially  a  mixture  of  camphor  and  nitrated  cellulose  from  cotton  or  paper. 
These  bodies  are  esters  and  therefore  may  be  decomposed  by  alkalies. 


CHAPTER  IV. 

THE  FATS  AND  SUBSTANCES  RELATED  TO  THEM. 

In  nature  we  find  a  large  number  of  esters  composed  of  the  fatty- 
acids  united  to  glyceryl.  These  are  the  ordinary  fats  and  as  foodstuffs 
they  are  nearly  as  important  as  the  carbohydrates.  In  structure  they 
are  practically  all  of  the  type  C3H5(C»H2re_102)3,  but  include  bodies 
of  widely  different  physical  properties.  Some  are  liquids,  while  others  at 
the  ordinary  temperature  are  hard  solids.  Nearly  all  vegetable  products 
contain  fats  of  some  kind;  often  the  amount  is  very  small,  but  fre- 
quently it  constitutes  fully  50  per  cent  by  weight  of  the  seed,  nut  or 
fruit  in  question.  In  the  animal  kingdom  fats  are  always  present, 
in  some  amount,  in  all  organisms.  The  animal  fats  are  often  derived 
from  the  vegetable  fats  consumed  as  food. 

THE    NATURAL    FATS. 

The  important  fatty  acids  combined  with  the  radical  of  glycerol, 
C3H5(OH)3,  are  given  in  the  following  table.  The  combinations  are 
essentially  like  that  illustrated  by  this  structural  formula  of  stearin: 

CH2—  0-QsH350 

CH  _0-ClsH350 

CH2  —  O  —  C]8H350 

SATURATED  ACIDS,  CnH2re02. 

Formic  acid,  HCHO,         "| 

Acetic  acid,  HC2H302  l-glycerides  not  natural  substances. 

Propionic  acid,  HC3H502  J 

Butyric  acid,  HQH702,  occurs  in  butter  fat  as  glyceride. 
Pentoic  acid,  HC5H902,.  valeric  acid  occurs  as  a  natural  compound. 
Caproic  acid,  HC6Hn02,  in  butter  fat  as  glyceride. 
(Enanthylic  acid,  HC7Hi302,  does  not  occur  as  glyceride. 
Caprylic  acid,  HCsH1502,  as  glyceride  in  butter  and  other  fats. 
Pelargonic  acid,  HC9H1702,  in  vegetable  kingdom,  but  not  as  glyceride. 
Capric  acid,  HQ„H]902,  in  butter  and  other  fats  as  glyceride. 
Undecylic  acid,  HC„H2102,  not  found  as  natural  glyceride. 
Laurie  acid,  HC12H2302,  as  glycerol  ester  in  several  fats. 
Myristic  acid,  HC14H2702,  in  nutmeg  butter  and  other  fats  as  glyceride. 
Palmitic  acid,  HC16H3102,  as  glyceride  in  many  fats. 
Margaric  acid,  HC17H3302  obtained  as  artificial  glyceride. 
Stearic  acid,  HCJSH3502,  as  glyceride  in  many  fats. 
Arachidic  acid,  HC20H39O2,  as  glyceride  in  peanut  oil. 
Behenic  acid,  HC22H4302„  as  glyceride  in  certain  oils. 

40 


THE    FATS    AND    SUBSTANCES    RELATED    TO    THEM.  4 1 

A  few  other  acids  of  this  series  are  known  in  glycerol  combina- 
tions but  they  are  unimportant. 

NON-SATURATED  ACIDS,  CnH2n-202  AND  CnH2n.402. 

Not  many  of  these  acids  occur  as  natural  glycerides. 

Hypogaeic  acid,  C16H30O2,  as  glyceride  in  peanut  oil. 
Oleic  acid,  CsH^Oo,  as  glyceride  in  many  oils. 
Linoleic  acid,  C1SH3202,  as  glyceride  in  drying  oils. 
Ricinoleic  acid,  dsH^Os,  as  glyceride  in  castor  oil. 

A  large  number  of  the  acids  in  the  first  list  and  two  in  the  second 
occur  in  the  edible  fats,  while  some  are  found  in  products  of  little 
importance,  or  in  such  as  have  technical  uses  only.  The  fats  which 
are  most  commonly  used  as  foods  are  those  consisting  largely  or 
wholly  of  stearin,  palmitin  and  olein.  Butter  fat,  however,  contains 
relatively  large  amounts  of  other  glycerides. 

Reactions  of  Fats.  Certain  general  reactions  are  common  to 
practically  all  fats  and  will  be  explained  here  in  detail. 

Saponification.     This  term  is  applied  to  the  change  which  fol- 
lows when  fats  are  treated  with  alkali  solutions,  usually  with  applica- 
tion of  heat.    The  fats  decompose  more  or  less  readily,  and  as  products 
we  have  soaps  and  glycerol,  according  to  these  equations : 
C3H5(C1SH3502)3  +  3KOH  =  C3H5(OH)3  +  3KC1SH3302 
2C3H5(CISH3302)3  +  3PbO  +  3H20  =  2C3H5(OH)3  +  3Pb(C18H3302)2 

In  the  first  case  potassium  stearate  is  formed  and  in  the  second 
lead  oleate,  which  is  the  important  constituent  of  lead  plaster.  Here 
lead  oxide  and  water  are  equivalent  to  lead  hydroxide.  Much  of  the 
glycerol  of  commerce  is  produced  by  such  decompositions.  When 
sodium  hydroxide  is  used  with  the  common  fats  ordinary  hard  soap 
results. 

An  analogous  change  occurs  when  fats  are  subjected  to  the  action 
of  water  at  a  high  temperature  or  superheated  steam.  We  have  here 
hydrolysis  purely,  although  the  term  saponification  by  steam  is  some- 
times applied.  The  same  reaction  is  brought  about  by  certain  enzymes 
at  the  ordinary  temperature,  for  example  by  the  enzyme  known  as 
lipase  or  steapsin  in  the  pancreatic  secretion.  This  will  be  discussed 
fully  later  as  it  is  important  in  the  digestion  of  fats.  Sometimes  the 
reaction  is  complete  as  shown  by  this  equation : 

C3Hr,rC,JI,,02)3  +  3H20  =  C3Hr,(OH)3  +  3CISH30O2 
But  products  of  partial  hydrolysis,   monostearin  and   distcarin,   for 
example,  may  be  left,  as : 

rC .n  <>■  ("OH 

C,llA  C,JT,,r,02  +  H20  =  C,l  I'M' ) ..  I-  HC«H„Oa 

Ic.j  Lc,j(  .'  >. 


42  PHYSIOLOGICAL    CHEMISTRY. 

ILLUSTRATIVE  TESTS. 

Some  of  the  saponifications  are  illustrated  by  these  experiments: 

Experiment.  Boil  about  25  gm.  of  tallow  with  a  solution  of  10  gm.  of  potas- 
sium hydroxide  in  100  cc.  of  water.  Stir  the  mixture  thoroughly  until  it  becomes 
homogeneous;  that  is,  until  no  oil  globules  are  seen  floating  on  top  of  the  aqueous 
liquid,  which  may  require  half  an  hour.  Add  water  from  time  to  time,  to  make 
up  for  that  lost  by  evaporation.  The  resulting  mass  is  a  mixture  of  glycerol, 
excess  of  alkali  and  soft  soap.  To  this  now  add  a  solution  of  15  gm.  of  common 
salt  in  75  cc.  of  water  and  heat  again,  which  brings  about  a  conversion  of  the 
soft  soap  into  the  hard  or  sodium  soap.  On  cooling,  this  separates  and  floats  on 
the  excess  of  spent  lye  and  salt  solution. 

Experiment.  The  presence  of  fatty  acids  in  the  above  soap  may  be  shown  by 
adding  to  a  portion  of  it  enough  hydrochloric  acid  to  decompose  the  soap.  Use 
about  half  the  product  of  the  experiment,  dilute  with  water,  and  add  the  acid 
in  slight  excess,  about  10  cc.  of  the  strong  commercial  acid.  Warm  on  the  water- 
bath,  which  will  cause  the  liberated  fatty  acids  to  collect  on  the  surface  as  a  liquid 
layer  as  soon  as  the  temperature  becomes  high  enough.  Add  more  water  and 
allow  the  whole  to  cool.  A  semi-solid  layer  of  fatty  acids  can  now  be  lifted  from 
the  surface  of  the  liquid.  The  hardness  of  the  mixed  acids  depends  on  the  fat 
taken  for  experiment.  Mutton  and  beef  tallows  yield  very  solid  acids ;  with  lard 
the  mass  is  softer,  while  with  some  oils  the  acids  do  not  solidify  at  all  at  the  ordi- 
nary temperature. 

Experiment.  Dissolve  a  small  portion  of  the  fatty  acids  in  warm  alcohol,  nearly 
to  saturation.     On  cooling,  the  acids  separate  in  crystalline  scales. 

Experiment.  The  presence  of  glycerol  as  one  of  the  products  formed  by  the 
saponification  of  fats  is  best  shown  as  follows : 

Mix  50  cc.  of  cottonseed  oil  with  25  gm.  of  litharge  and  100  cc.  of  water  in  a 
porcelain  dish.  Place  over  a  Bunsen  burner  on  gauze  and  stir  until  all  oil 
globules  have  disappeared,  adding  a  little  water  from  time  to  time.  The  litharge 
with  water  acts  as  lead  hydroxide  and  saponifies  the  fat,  forming  an  insoluble  lead 
soap,  or  plaster,  and  glycerol.  When  the  saponification  is  complete  add  more  water, 
heat  and  stir  well  to  dissolve  glycerol.  Allow  to  settle  a  short  time  and  pour 
the  aqueous  solution  through  a  filter.  To  the  residue  add  water  again,  heat,  allow 
to  settle  and  pour  through  the  same  filter.  Concentrate  the  mixed  filtrates  to  a 
small  volume  and  after  cooling  observe  the  sweet  taste  of  the  thickish  residue. 

Experiment.  Dissolve  a  portion  of  the  sodium  soap  in  water  with  aid  of  a  little 
alcohol.  Then  add  some  solution  of  calcium  chloride  or  lead  acetate.  A  white 
precipitate  is  formed,  as  the  calcium  and  lead  salts  of  the  fatty  acids  are  not 
soluble  in  water.  Hard  waters,  which  contain  salts  of  calcium  and  magnesium, 
decompose  soap  in  the  same  manner. 

Other  Reactions.  The  common  fats  are  insoluble  in  water  and 
when  mixed  with  the  latter  tend  to  separate  immediately.  However, 
it  is  possible  to  convert  the  fats  and  water  into  a  peculiar  mixture 
called  an  emulsion  which  does  not  separate  into  two  layers  on  standing. 
In  this  condition  the  fat  consists  of  extremely  minute  globules  which 
remain  in  suspension  and  which  may  be  passed  through  the  pores  of 
coarse  filter  paper.  It  does  not  seem  possible  to  secure  an  emulsion 
with  perfectly  neutral  fats,  and  in  most  cases  the  phenomenon  de- 
pends on  the  presence  of  a  trace  of  soap  formed.    In  the  processes  of 


THE    FATS    AND    SUBSTANCES    RELATED    TO    THEM. 


43 


digestion  of  fats  in  the  animal  body  emulsification  plays  a  very  im- 
portant part  as  will  be  shown  later.  It  follows,  probably,  the  partial 
hydrolysis  of  the  fats  by  lipase,  referred  to  above. 

As  they  exist  in  the  animal  or  vegetable  organism  the  fats  are 
doubtless  all  amorphous  substances,  but  in  the  separated  condition  the 
solid    fats   always   become   more   or   less   crystalline.      This   may   be 


Fig.  4.     Mutton  tallow  crystallized  fror 
chloroform.      300  diameters. 


Fig.  5.     Cat  fat  crystallized  from  chlo- 
roform.    300  diameters. 


Fig.   6.     Beef   tallow   crystallized   from 
chloroform.      300  diameters. 


Fig.    7.      Beef   tallow    crystallized    from 
chloroform.      300  diameters. 


readily  shown  by  dissolving  some  fat  in  a  proper  solvent  which  is 
then  allowed  to  evaporate  slowly. 

The  common  fats  can  not  be  distilled  under  the  ordinary  pressure 
without  decomposition,  and  the  distillation  of  the  fatty  acids  is  also 
difficult.  When  the  common  fats  are  strongly  heated  they  emit  a  pe- 
culiar odor,  due  to  the  acrolein  formed  by  the  partial  destruction  of 
glycerol.  The  following  experiments  illustrate  some  of  the  points 
referred  to : 


44  PHYSIOLOGICAL    CHEMISTRY. 

Experiment.  Note  the  solubility  of  small  bits  of  tallow  in  ether,  chloroform, 
benzine  and  alcohol,  using  in  each  case  the  same  volume  of  liquid,  with  equal 
weights  of  fat.  The  solubility  in  alcohol  will  be  found  much  less  than  in  the 
other  menstrua. 

Experiment.  Dissolve  some  mutton  or  beef  tallow  in  chloroform  and  with  a 
glass  rod  put  two  or  three  drops  of  the  nearly  saturated  solution  on  the  center  of 
a  glass  slide.  As  the  chloroform  evaporates  a  film  begins  to  form  on  the  top  of 
the  drop.  Now  put  on  a  perfectly  clean  cover  glass  and  allow  to  stand  until 
crystallization  is  complete,  which  may  require  only  a  few  minutes  or  some  hours, 
the  time  necessary  depending  on  the  temperature  and  on  the  concentration  of  the 
solution.  Examine  the  crystals  with  a  microscope.  Use  a  power  of  200  to  300 
diameters.  By  repeating  the  experiment  with  different  fats  considerable  variation 
in  the  form  of  the  crystals  may  be  noticed,  which  is  shown  in  the  annexed  cuts. 

Experiment.  Add  to  5  cc.  of  cottonseed  oil  half  its  volume  of  strong  white  of 
egg  solution  and  shake  thoroughly.  The  liquids  mix  and  form  a  white  mass  or 
emulsion  which,  however,  is  not  usually  stable. 

Experiment.  To  5  cc.  of  cottonseed  oil  containing  a  little  free  fatty  acid  add  10 
drops  of  strong  sodium  carbonate  solution  and  shake.  A  good  stable  emulsion  is 
made  in  this  way,  as  the  sodium  of  the  alkali  solution  forms  a  soap  with  the  free 
acid  and  this  appears  to  form  a  film  around  the  little  fat  globules  which  prevents 
their  flowing  together  again. 

Origin  of  Fats  in  the  Body.  The  question  of  the  formation  of 
fats  in  the  animal  organism  has  been  much  discussed.  It  was  once 
assumed  that,  like  protein  substances,  the  fats  are  products  of  the 
vegetable  world  only,  and  that  the  animal  has  not  the  power  of  build- 
ing them  up  from  compounds  which  are  not  fats.  But  this  view  is 
not  correct,  as  we  have  abundant  proof  that  fats  may  be  made  in 
other  ways.  Much  has  been  learned  from  the  results  of  cattle  feeding 
experiments  carried  out  in  agricultural  experiment  stations,  where 
the  gain  in  fat  is  often  much  in  excess  of  what  could  be  accounted  for 
by  the  amount  of  fats  in  the  food  consumed.  This  gain  must  in  some 
way  be  due  to  the  effect  of  the  carbohydrate  and  protein  substances 
in  the  rations  fed.  The  .fattening  power  of  sugar  has  long  been 
recognized,  but  this  has  been  in  part  accounted  for  on  the  theory  that 
the  sugar  acts  to  protect  the  fats  of  the  body  from  oxidation,  by  being 
readily  oxidized  itself  to  keep  up  the  body  energy.  But  much  evi- 
dence has  been  accumulated  to  show  that  carbohydrates  take  part 
directly  in  the  production  of  fats.  How  this  is  accomplished  is  not 
known,  but  in  the  processes  syntheses  and  oxidations  both  must  be 
concerned,  since  the  fat  molecules  are  more  complex  than  those  of  the 
carbohydrates. 

It  is  further  likely  that  protein  compounds  are  important  factors 
in  fat  production.  Many  writers  have  indeed  assumed  that  we  have  in 
the  breaking  down  of  protein  molecules  the  chief  sources  of  fats,  but 
this  view  has  been  strongly  combated.     Indirectly  the  transformation 


THE    FATS    AND    SUBSTANCES    RELATED    TO    THEM.  45 

may  follow  in  this  way :  It  is  known  that  sugars  are  formed  as  cleav- 
age products  of  certain  albumins  in  the  ordinary  katabolic  processes  of 
the  body  and  possibly  a  portion  of  the  sugars  thus  formed  may  be 
then  built  up  to  produce  fats. 

The  existence  of  the  substance  known  as  adipocere  has  long  been 
held  to  furnish  a  pretty  strong  proof  of  the  production  of  fatty  acids 
from  protein.  This  adipocere  or  cadaver  wax  is  often  found  in  large 
masses  in  old  cemeteries  and  consists  of  fatty  acids,  calcium  and  am- 
monium soaps  in  the  main.  It  is  held  that  this  substance  could  not 
possibly  have  come  from  the  small  amounts  of  fat  ordinarily  present 
in  cadavers  but  must  have  been  produced  from  the  muscular  portions 
of  the  body.  This  view  has  met  with  objections,  however,  and  at- 
tempts are  made  to  account  for  the  development  of  the  adipocere  in 
other  ways. 

In  the  body  fats  constitute  a  reserve  material  in  which  potential 
energy  is  conveniently  stored  up.  In  sickness  or  in  wasting  diseases 
where  there  is  a  partial  or  complete  failure  in  nutrition  this  fat  is 
called  upon  to  supply  the  needs  of  the  body.  The  fats  are  oxidized 
while  the  muscular  tissue  is  preserved. 

PHYSIOLOGICALLY  IMPORTANT  FATS. 

Stearin  or  Tristearin,  C3H5(C18H3502)3.  This  is  a  simple  fat 
which  does  not  occur  in  nature  unmixed  with  other  fats.  When  sepa- 
rated in  purest  possible  condition  it  shows  a  melting  point  of  550  to 
580.  It  is  the  hardest  of  the  common  simple  fats  and  apparently  the 
least  soluble  in  alcohol  or  ether.  It  may  be  separated  in  the  form  of 
rectangular  plates  by  crystallizing  from  hot  alcohol.  Stearic  acid 
may  be  obtained  in  the  form  of  pearly  crystalline  plates  or  scales.  It 
melts  at  71  °. 

Palmitin  or  Tripalmitin,  C3H5(C16H3102)3.  The  perfect  sepa- 
ration of  this  fat  from  stearin,  with  which  it  is  usually  associated, 
is  a  matter  of  considerable  difficulty.  The  fats  are  much  alike. 
The  melting  point  is  variously  stated  by  different  observers,  but 
appears  to  be  about  510.  A  mixture  of  stearin  and  palmitin  was 
formerly  supposed  to  be  a  distinct  fat  and  was  called  margarin, 
C3H5('Ci7H3302)3.  This  fat  has  been  produced  artificially  but  it 
does  not  appear  to  be  a  natural  product.  Palmitic  acid  resembles 
stearic  acid  in  appearance  and  solubility;  both  acids  are  slowly  soluble 
in  strong  hot  alcohol  and  yield  crystalline  plates  on  cooling.  The 
melting  point  of  palmitic  acid  is  about  620. 

Olein  or  Triolein,  C3H5(C18H3302);!.    This  is  a  liquid  fat  at  the 


46  PHYSIOLOGICAL    CHEMISTRY. 

ordinary  temperature  and  is  a  constituent  of  most  of  the  natural  fats 
and  oils.  Some  fatty  oils  are  nearly  pure  olein  and  become  solid  at  a 
low  temperature.  The  soft  consistence  of  lard,  human  fat  and  sev- 
eral other  natural  mixtures  is  due  to  the  olein  present.  Olein  is  a 
nonsaturated  fat  and  will  therefore  show  an  absorption  coefficient 
as  explained  below.  By  the  action  of  reagents  yielding  nitrous  acid 
it  is  converted  into  an  isomeric  substance  known  as  elaidin. 

Oleic  acid  in  pure  condition  is  not  very  stable,  because  of  its  un- 
saturated structure.  It  is  an  oily  liquid  at  the  ordinary  temperature, 
but  below  140  is  converted  into  a  crystalline  solid.  By  treatment 
with  nitrous  acid  it  yields  the  isomeric  elaidic  acid.  Oleic  acid  is 
characterized  by  forming  a  lead  salt  which  is  soluble  in  ether  while 
lead  palmitate  and  stearate  are  practically  insoluble. 

Lard  and  Tallow  are  essentially  mixtures  of  the  three  fats,  pal- 
mitin,  stearin  and  olein.  By  heating  lard  to  its  melting  point,  cooling 
slowly  and  subjecting  the  warm  mass  to  pressure  in  a  filter  press  the 
softer  portion,  consisting  mainly  of  olein,  may  be  separated.  This  is 
known  as  lard  oil,  while  the  harder  residue  is  sometimes  called  lard 
stearin.  It  has  about  the  consistence  of  butter.  By  subjecting  beef 
suet  to  the  same  treatment  a  soft  portion  known  as  oleo  oil  is  separated 
from  a  solid  residue  called  beef  stearin.  The  oleo  oil  is  the  material 
most  often  employed  under  the  name  of  oleomargarin  as  a  substitute 
for  butter.  A  mixture  of  somewhat  similar  consistence  is  made  in 
other  ways ;  for  example  by  combining  cottonseed  oil  with  beef  stearin. 

Oleomargarin  is  the  name  given  by  law  to  these  butter  substitutes 
in  the  United  States.  Sometimes  the  fats  are  churned  with  milk  or 
mixed  with  a  certain  amount  of  real  butter  to  furnish  a  product  with 
flavor  suggesting  butter.  The  name  butterine  is  usually  given  to 
such  mixtures  and  when  properly  made  they  are  wholesome  and  in 
every  way  as  good  as  butter  from  the  standpoint  of  nutritive  value. 

Butter.  The  fat  of  milk  is  a  very  complex  mixture  and  an  exact 
separation  has  not  yet  been  made  by  the  methods  of  chemical  analysis. 
According  to  the  older  notion  butter  fat  contains  essentially  olein, 
stearin  and  palmitin,  with  a  little  butyrin,  to  which  the  flavor  and  odor 
are  largely  due,  but  it  has  been  shown  that  other  glycerol  esters  are 
certainly  present.  The  results  of  some  recent  examinations  may  be 
approximately  expressed  as  follows : 

Glyceryl   butyrate    7.0 

Glyceryl  caproate,  caprylate  and  caprate    2.0 

Glyceryl  oleate  36.0 

Glyceryl  myristate,  palmitate  and  stearate   55.0 

100.0 


THE    FATS    AND    SUBSTANCES    RELATED    TO    THEM.  47 

From  various  investigations  it  appears  that  these  fats  are  not 
present  as  simple  esters,  but  may  possibly  exist  in  combinations  repre- 
sented by  formulas  like  this : 

rC18H3502 
C3Ha<  CieH3I02 
I  QH702 

The  melting  point  of  butter  fat  is  between  38 °  and  45 °.  On  melting 
100  parts  by  weight  of  pure  butter  fat,  saponifying,  separating  the 
fatty  acids  and  washing  out  everything  soluble  in  hot  water  (butyric 
acid,  etc.)  it  is  found  that  the  insoluble  residue  left  amounts  in  the 
mean  to  88  per  cent,  but  may  be  more  or  less  with  different  grades  of 
butter.  Commercial  butter  contains  in  the  mean  about  85  per  cent 
of  fat,  10  per  cent  of  water  and  5  per  cent  of  salt. 

Human  Fat.  This  consists  essentially  of  olein,  palmitin  and 
stearin.  In  the  fat  of  children  the  solid  glycerides  apparently  are  in 
excess,  while  in  later  life  the  proportion  of  olein  increases,  so  that  the 
separated  fat  may  appear  quite  soft.  In  the  human  adult  the  olein 
may  amount  to  75  per  cent  of  the  whole  fat,  but  the  proportion  varies 
with  different  parts  of  the  body. 

Glycerol.  Since  it  is  a  constituent  of  all  the  true  fats  a  few  words 
about  this  alcohol  will  be  in  order  here.  The  substance  was  first  recog- 
nized in  the  aqueous  liquid  left  in  the  preparation  of  lead  plaster  and 
for  many  years  all  used  in  pharmacy  and  in  the  manufacture  of  cos- 
metics was  made  by  the  same  reactions.  Since  its  importance  in 
technology  was  recognized  it  has  been  produced  on  the  large  scale  by 
other  kinds  of  saponification  or  hydrolysis.  In  pure  condition  it  is  a 
thick,  sweetish  liquid  with  a  specific  gravity  of  1.266  at  150,  referred 
to  water  at  the  same  temperature.  It  mixes  with  water  and  alcohol 
in  all  proportions,  but  is  not  soluble  in  pure  chloroform,  benzene,  car- 
bon disulphide  or  petroleum  ether.  It  is  very  slightly  soluble  in  ether. 
Like  other  alcohols  it  may  be  combined  with  acids  to  form  esters. 
With  stearic  acid  mono-,  di-  and  tri-stearin  are  produced,  the  last 
being  identical  with  the  natural  fat.  When  fed  to  animals  glycerol 
may  be  oxidized  to  a  limited  extent  only.  Any  excess  of  it  fails  to  be 
assimilated  and  soon  produces  disorders  in  digestion  and  absorption. 
Its  food  value,  in  free  form,  is  therefore  very  slight. 

Recognition  and  Determination  of  Fats.  In  physiological  chemical  investiga- 
tions fats  are  separated  from  accompanying  substances  through  their  solubility  in 
warm  ether,  chloroform  or  petroleum  ether.  The  carbohydrates,  protein  bodies 
and  salts  are  not  soluble  in  these  liquids.  The  saponification  test  is  also  of  value  in 
identification.  Many  of  the  fats  contain  unsaturated  acid  groups  and  are  there- 
fore able  to  absorb  certain  amounts  of  halogens  from  specially  prepared  solutions. 


48  PHYSIOLOGICAL    CHEMISTRY. 

Oleic  acid,  C^H^O^  absorbs  iodine  or  bromine  to  form  C^H^IsOu  or  dsH^BrjOj. 
The  fat  to  be  examined  is  dissolved  in  chloroform  and  treated  with  the  standard 
solution.  After  a  time  the  excess  of  iodine  (or  bromine)  is  determined,  and  that 
absorbed  by  the  fat  is  a  measure  of  the  non-saturated  acid  present.  Linoleic  acid 
absorbs  twice  as  much  iodine  or  bromine  as  oleic  acid,  as  the  formula  C1SH3202 
becomes  C1SH32I402. 

The  determination  of  the  amount  of  insoluble  fatty  acids  furnished  by  a  given 
weight  of  fat  is  also  a  valuable  factor  in  the  study  of  these  bodies.  In  another 
method  the  fat  is  saponified,  the  soap  formed  decomposed  by  dilute  sulphuric  acid 
and  the  resultant  product  subjected  to  distillation.  Fatty  acids  which  are  volatile 
pass  over  and  collect  in  the  distillate,  where  their  amount  may  be  determined  in 
terms  of  KOH  or  NaOH,  by  titration.  In  this  treatment  stearin  and  palmitin 
yield  acids  not  volatile  with  steam.  This  is  a  valuable  test  and  is  applied  in  the 
examination  of  butter  supposed  to  be  adulterated  with  other  fats. 

In  certain  lines  of  investigation  and  especially  in  the  examination  of  tissues  by 
aid  of  the  microscope,  fat  is  recognized  by  its  coloration  through  the  coaltar  product 
known  as  Sudan  III.  This  is  one  of  the  few  colors  which  are  soluble  in  fats 
and  fatty  oils.     A  yellowish-red  color  is  imparted. 

Lecithin.  This  is  a  peculiar  complex  body  which  contains  phos- 
phoric acid  and  an  organic  basic  group  in  place  of  one  of  the  fatty 
acid  radicals  in  the  common  fats.  It  is  found  in  the  vegetable  king- 
dom, but  commonly  and  most  characteristically  in  many  animal  tis- 
sues, in  the  brain  and  nerve  tissue,  blood,  lymph,  milk,  pus,  yolk  of 
egg,  etc.  It  is  most  readily  prepared  from  the  last  named  substance. 
The  following  formula  represents  the  supposed  structure  of  the  body, 


-_0-C18H350 
C3)\,A  -0-C18H350^ 

'  OH 


—  O  —  PO-HO-0-C2H4  —  N|  c- 


in  which  distearylglycero-phosphoric  acid  is  combined  with  the  base 
choline : 

(CH3)3  =  N  {QH4oH 

It  appears  that  several  forms  of  lecithin  exist,  containing  oleic  and 
palmitic  acid  as  well  as  stearic.  They  undergo  saponification,  yielding 
fatty  acids,  glycero-phosphoric  acid  and  choline.  They  are  soluble 
in  ether  and  alcohol  and  in  other  respects  resemble  the  true  fats.  With 
water  lecithin  swells  to  a  gelatinous  mass  which  under  the  microscope 
presents  a  characteristic  appearance.  The  function  of  lecithin  in  the 
body  is  not  understood,  but  from  the  fact  of  its  wide  distribution  and 
its  occurrence  in  milk  it  is  reasonable  to  assume  that  it  performs  some 
important  part. 

The  above  formula  represents  the  simple  or  typical  lecithin.  As  recent  investiga- 
tions have  shown  that  a  number  of  related  bodies  exist  containing  other  propor- 
tions of  nitrogen  and  phosphorus  it  has  been  proposed  to  give  the  name  phos- 
phatides to  the  whole  group.    The  group  name  lecithan  is  also  used.    The  chemistry 


THE    FATS    AND    SUBSTANCES    RELATED    TO    THEM.  49 

of  these  bodies  is  far  from  simple.  Some  of  them  appear  to  be  associated  with 
sugars,  and  others  with  proteins  in  the  animal  and  vegetable  tissues,  but  as  they 
suffer  decomposition  very  easily  their  separation  in  pure  form  for  study  is  an 
extremely  difficult  problem. 

The  Waxes.  These  bodies  bear  some  resemblance  to  the  fats  and 
will  be  briefly  mentioned  here.  They  consist  largely  of  esters  of 
the  higher  monohydric  alcohols  of  the  saturated  series,  and  in 
most  cases  are  complex  mixtures  of  which  the  composition  is  not 
exactly  known.  Spermaceti  seems  to  consist  largely  of  cetyl  palmitate, 
C16H33OC16H310.  Beeswax  contains  some  free  acid,  cerotic  acid,  in 
addition  to  the  esters.  The  most  important  constituent  is  apparently 
myricin  or  myricyl  palmitate,  C30H61OC16H31O.  The  waxes  are  not 
easily  saponified  and  as  a  rule  clear  soap  solutions  are  not  obtained. 

Cholesterol.  This  substance  is  an  alcohol,  but  in  appearance  it 
resembles  some  of  the  solid  fats  and  is  associated  with  them  in  several 
natural  products.  Hence  it  is  in  place  to  describe  it  here.  The  for- 
mula C27H45OH  probably  represents  the  composition  of  the  body 
which  is  found  in  the  brain,  yolk  of  egg,  the  liver,  blood  and  other 
tissues  and  is  especially  abundant  in  the  fat  of  wool.  It  constitutes 
also  the  main  substance  in  certain  gall-stones  from  which  it  may  be 
separated  in  nearly  pure  condition.  In  wool  fat  it  exists  partly  in  the 
free  state  and  partly  in  combination  with  fatty  acids  in  the  form  of 
esters. 

It  is  readily  soluble  in  hot  alcohol,  ether,  benzene  and  chloroform, 
but  not  in  water  or  alkali  solutions.  It  is  therefore  left  as  an  insoluble 
residue  in  the  saponification  of  fatty  mixtures  containing  it.  Under 
the  microscope  pure  cholesterol  appears  as  a  mass  of  white  plates  with 
sharp  angles.  The  cholesterol  esters  combine  with  water  to  form 
stable  emulsions,  and  it  is  probably  on  account  of  the  presence  of  these 
esters  that  the  substance  known  as  lanolin  is  practically  valuable.  This 
lanolin  is  made  from  purified  wool  fat  and  is  largely  used  in  the  prepa- 
ration of  salves  and  ointments. 

An  isomeric  substance  known  as  isocholesterol  is  often  found  asso- 
ciated with  the  true  cholesterol,  especially  in  wool  fat.  In  the  vege- 
table kingdom  other  forms  of  cholesterol  are  widely  distributed  in 
small  quantities,  being  found  in  most  oils  and  seeds.  All  forms  of 
cholesterol  have  a  marked  action  on  polarized  light. 

Experiment.  If  gall-stones  are  obtainable  the  following  reactions  may  be  car- 
ried out  in  illustration  of  properties  of  cholesterol.  Crush  the  stones  to  a  powder 
and  boil  with  water  to  remove  anything  soluble.  Extract  the  residue  several  times 
with  hot  alcohol,  filter,  unite  the  solutions  and  allow  the  cholesterol  to  crystallize 
on  cooling.     As  some  fat  may  be  present  this  must  be  removed  by  saponifying  with 

5 


50  PHYSIOLOGICAL    CHEMISTRY. 

a  little  alcoholic  potassa  solution.  After  saponification  boil  off  the  alcohol  and 
extract  the  dry  residue  with  ether,  in  which  soaps  are  insoluble.  On  evaporation 
of  the  ether  a  nearly  pure  cholesterol  is  obtained.  It  may  be  further  purified  by 
recrystallization  from  hot  alcohol.     With  the  substance  these  tests  may  be  made: 

Salkowski's  Test.  Dissolve  about  10  milligrams  of  cholesterol  in  2  cubic  centi- 
meters of  chloroform  and  shake  with  an  equal  volume  of  strong  sulphuric  acid. 
The  chloroform  becomes  colored  blood  red,  then  cherry  red  and  finally  purple. 
The  acid  shows  a  dark  green  fluorescence.  If  the  chloroform  is  poured  into  a  dish 
the  color  changes  to  blue,  then  green  and  finally  yellow. 

Burchard-Liebermann  Test.  Dissolve  about  10  milligrams  of  cholesterol  in  2 
cubic  centimeters  of  chloroform,  add  20  drops  of  acetic  anhydride  and  1  drop  of 
strong  sulphuric  acid.     A  violet  pink  color  results. 

The  appearance  of  cholesterol  plates  should  be  studied  under  the  microscope. 

The  presence  of  cholesterol  in  the  ester  form  in  lanolin  and  similar 
preparations  of  wool  fat  may  be  shown  by  the  above  tests. 


CHAPTER     V. 
THE    PROTEIN    SUBSTANCES. 

These  extremely  important  bodies,  usually  called  albuminous  bodies, 
are  found  in  vegetable  and  animal  organisms  of  all  kinds,  and  in  some 
form  are  essential  elements  in  cell  growth.  Unlike  the  fats  and  carbo- 
hydrates, they  seem  to  be  elaborated  in  the  vegetable  kingdom  only; 
or,  at  any  rate,  the  fundamental  structures  in  them  appear  to  be  formed 
in  vegetable  growth  only.  The  animal  is  able  to  modify  and  trans- 
form to  some  extent,  but  apparently  can  not  build  them  up  from 
simple  materials.  In  composition  the  protein  bodies  are  extremely 
complex;  qualitatively  they  contain  carbon,  hydrogen,  oxygen,  nitro- 
gen and  sulphur.  In  an  important  group  of  these  bodies  phosphorus 
is  also  present,  and  a  few  contain  iron.  The  quantitative  composition 
of  some  of  the  best  known  protein  compounds  is  expressed  approxi- 
mately as  follows : 

Per   Cent. 

C  50.0-55.0 

H  6.5-  7-3 

O  19.0-23.0 

N  15.0-17.0 

S  0.3-  2.4 

Attempts  have  been  made  to  calculate  formulas  for  certain  protein 
bodies  from  the  results  of  analyses,  but  no  great  importance  attaches 
to  the  empirical  formulas  so  reached.  The  best  analyses  made  of  the 
compounds  differ  among  themselves  to  an  extent  that  makes  a  definite 
result  quite  impossible.  This  is  largely  due  to  the  fact  that  there  are 
great  practical  difficulties  in  the  way  of  properly  purifying  the  sub- 
stances as  a  preliminary  to  analysis ;  they  all  occur  mixed  with  other 
compounds,  such  as  fats,  carbohydrates  and  mineral  matters,  and  to 
remove  these  without  in  any  way  altering  the  composition  of  the 
complex  albuminous  molecule  is  extremely  difficult,  if  not  impossible. 
The  formulas  which  have  been  published  are  interesting  chiefly  in 
showing  roughly  how  complex  the  structures  certainly  are.  For  serum 
albumin  TTofmeister  has  given  this  minimum  formula: 

V^4BoH  J20-N  116^5»^-'n0! 

while  for  t^  albumin  he  has  given  this: 

51 


52  PHYSIOLOGICAL    CHEMISTRY. 

Even  more  complex  formulas  are  given,  for  example  this : 

C755.H.1215JN  195  "10*-'  235- 

These  formulas  are  in  a  measure  based  on  an  assumption  as  to  the 
number  of  sulphur  atoms  present,  about  which  something  will  be  said 
later. 

The  usual  methods  of  fixing  organic  formulas  by  aid  of  a  molecular 
weight  determination  can  not  be  successfully  applied  in  these  cases. 
In  the  cryoscopic  method,  for  example,  the  traces  of  mineral  im- 
purities present  have  possibly  as  much  influence  on  the  result  as  the 
whole  weight  of  dry  protein.  Because  of  changes  in  composition  at  a 
high  temperature  the  boiling  point  method,  even  if  otherwise  reliable, 
can  not  be  applied,  and  methods  based  on  osmotic  pressure  observations 
lead  to  results  of  no  value. 

CLASSIFICATION    OF    THE    PROTEIN    BODIES. 

The  substances  thus  far  studied  have  been  divided  into  groups  or 
classes  dependent  on  chemical  composition  or  structure.  With  the 
protein  compounds  this  is  only  partially  possible  because  of  our  lack 
of  full  knowledge  in  this  direction.  Of  the  structural  relations  of  the 
protein  molecules  nothing  whatever  is  known,  while  of  composition 
only  a  few  general  facts  are  clearly  enough  established  to  be  available 
in  a  scheme  of  classification.  The  first  efforts  at  classification,  which 
we  owe  largely  to  the  work  of  Hoppe-Seyler,  were  therefore  essen- 
tially empirical.  Other  schemes  were  later  proposed  as  more  facts 
were  brought  to  light,  so  that  finally  a  grouping  like  the  following 
came  to  be  gradually  accepted  by  physiological  chemists,  with  slight 
differences  in  details  only.  The  arrangement  below  is  that  of  Ham- 
marsten,  in  the  form  given  by  Cohnheim.  He  makes  four  principal 
divisions  as  follows : 


Protein  Bodies 


'  True  or  Native  Albumins. 
Derived  Albumins  or  Transformation 

Products. 
Proteids. 
Albumoids. 


THE    PROTEIN    SUBSTANCES. 


53 


These  four  great  divisions  may  be  further  subdivided : 

Albumins  proper. 

Serum  albumin,  egg  albumin,  lactalbumin. 
Globulins. 

Serum    globulin,    egg    globulin,    lactoglobulin,    cell 
globulin,  vegetable  globulin. 
Coagulating  albumins. 

Fibrinogen,   myosin,  gluten  protein. 
Nucleoalbumins. 

Casein,    vitellins,    mucin-like    nucleoalbumins. 
Histones. 

Scomber-histone,  salmo-histone. 
Protamines. 

Salmin,  clupein,  sturin,   scombrin. 


TRUE   OR  NATIVE 
ALBUMINS. 


DERIVED    OR         f  Coagulated  or  Modified  Albumin. 
TRANSFORMATION  J    Acid  and  Alkali  Albumins,  Albuminates. 
PRODUCTS.  [  Albumoses,  Peptones. 


PROTEIDS. 


Nucleoproteids. 

Nucleic  acid  with  histone,  protamine,  etc. 
Hemoglobins. 

Hematin   with  histone. 
Glucoproteids. 

Combination  of  a  protein  and  carbohydrate  group, 
mucin,  mucoids,  phosphoglucoproteids. 
Lecithoproteids. 

Combination  of  a  protein  with  a  lecithin  body. 


ALBUMOIDS. 


Collagen,  forming  gelatin,  glue. 
Keratin,  in  horn,  hair,  nails,  etc. 
Elastin,  elastic  tissue. 
Amyloid,  in  pathological  formations. 
.  Spongin,  in  sponge. 


In  recent  years  enormous  additions  have  been  made  to  the  literature 
of  the  proteins,  and  many  new  substances  have  been  described.  Our 
knowledge  of  certain  groups  has  been  advanced  largely  through  the 
labors  of  the  Yale  school  of  chemists,  and  especially  by  Chittenden 
and  Osborne.  Much  of  our  systematic  knowledge  of  vegetable  pro- 
teins must  be  credited  to  these  investigators.  In  view  of  these  im- 
portant extensions  of  our  acquaintance  with  the  details  of  protein 
chemistry  a  committee  representing  the  American  Society  of  Bio- 
logical Chemists  and  the  American  Physiological  Society  has  recom- 
mended the  following  classification  of  the  bodies  in  question.  The 
known  substances  are  thrown  into  three  main  groups  in  place  of  four, 
as  above.    The  term  proteid  is  abandoned. 


54 


PHYSIOLOGICAL    CHEMISTRY. 


SIMPLE  PROTEINS 


CONJUGATED  PROTEINS. 


'  Albumins. 
Globulins. 
Glutelins. 
-   Alcohol-soluble  Proteins. 
Albumoids. 
Histones. 
Protamines. 

Nucleoproteins. 

Glycoproteins. 

Phosphoproteins. 

Hemoglobins. 

Lecithoproteins. 


DERIVED  PROTEINS. 


Primary  Derivatives. 


f  Proteans. 
-j  Metaproteins. 
[_  Coagulated  proteins. 


f  Proteoses. 
Secondary  Derivatives.  <  Peptones. 
(^  Peptides. 

The  differences  in  the  two  classifications  are  not  great.  The  albu- 
moids are  here  considered  as  simple  proteins,  which  is  probably  an 
advantage.  The  term  phospho  protein  in  the  new  classification  covers 
substances  like  the  nude o albumins  of  the  old. 


GENERAL  REACTIONS  OF  THE  PROTEINS. 

The  various  substances  in  the  protein  group  respond  to  a  number 
of  reactions  which,  taken  together,  are  sufficient  to  characterize  and 
identify  the  bodies  in  question.  They  all  contain  nitrogen  in  a  form 
to  be  liberated  as  ammonia  when  the  dry  substance  is  heated  with 
soda-lime.  A  positive  result  with  this  test  does  not,  of  course,  prove 
the  presence  of  a  protein  compound,  since  all  ammonium  salts  and 
amino  compounds  in  general  respond  to  it;  but  with  a  negative  result 
proteins  as  well  as  these  other  compounds  are  certainly  excluded. 
The  reaction  therefore  serves  as  a  preliminary  test  in  the  examina- 
tion of  unknown  substances  for  proteins.  The  test  may  be  easily 
carried  out  and  is  delicate. 

Experiment.  Mix  some  dried  albumin  or  some  wheat  flour  with  an  equal  bulk 
of  soda-lime  in  a  dry  test-tube.  Apply  heat  and  note  the  escape  of  ammoniacal 
vapors  as  shown  by  the  odor,  or  reaction  on  moist  litmus  paper.  The  fixed  alkali 
decomposes  the  protein  matter  very  quickly,  and  ammonia  always  results. 

COAGULATION  TESTS. 

Many  of  the  protein  substances  undergo  a  peculiar  change  known 
as  coagulation  when  heated,  or  treated  with  certain  reagents.  The 
test  is  characteristic  of  most  of  the  bodies  except  some  of  the  products 


THE    PROTEIN    SUBSTANCES.  5  5 

of  transformation.  This  coagulation  is  usually  accompanied  by  pre- 
cipitation, that  is,  the  body  is  thrown  out  of  solution  and  as  a  rule 
can  not  be  restored  to  its  original  condition.  But  there  are  cases  of 
precipitation  without  coagulation;  the  terms  must  not,  therefore,  be 
used  as  synonymous.  In  coagulation  proper  the  protein  body  becomes 
permanently  altered,  so  that  it  can  not  be  brought  into  its  original 
condition  again  by  addition  of  reagents  or  by  other  means.  On  the 
other  hand,  it  is  in  many  cases  possible  to  throw  a  protein  body  out  of 
solution  by  simply  adding  an  excess  of  some  inorganic  salt,  without 
at  the  same  time  producing  any  decided  alteration  in  the  character  of 
the  protein  precipitate.  By  largely  diluting  with  water  the  precipitate 
may  be  brought  into  the  soluble  condition  again.  This  will  be  illus- 
trated later  by  the  use  of  ammonium  sulphate  which  behaves  in  a 
characteristic  manner  with  different  proteins.  Many  of  these  have 
definite  precipitation  limits  with  the  sulphate.  That  is,  they  begin 
to  separate  when  the  amount  of  the  salt  reaches  a  certain  value,  and 
precipitate  completely  with  a  greater  concentration. 

Experiment.  The  simplest  coagulation  may  be  shown  by  boiling  a  dilute  white 
of  egg  solution.  As  long  as  it  is  perfectly  neutral  coagulation  follows  at  once, 
but  in  presence  of  alkali  acid  must  be  added  to  the  point  of  neutrality.  This 
behavior  finds  practical  application  in  the  ordinary  test  for  serum  albumin  as  it 
occurs  pathologically  in  urine.  The  precipitate  may  be  redissolved  only  by  some 
digestion  or  chemical  process  which  produces  a  new  substance. 

Coagulation  by  Reagents.  By  the  addition  of  certain  chemicals 
many  of  the  protein  compounds  are  easily  thrown  into  the  coagulated 
condition.  Some  of  the  most  characteristic  reactions  in  this  direction 
are  shown  by  simple  experiments  with  mineral  acids,  salts  of  heavy 
metals  and  the  so-called  alkaloid  reagents. 

Experiment.  Among  the  acids  which  bring  about  coagulation,  nitric  acid  is  the 
most  certain  in  its  action  and  is  commonly  used  in  practical  cases  where  it  is 
desired  to  recognize  a  small  amount  of  serum  or  egg  albumin  in  solution.  The 
test  may  be  made  by  adding  about  a  cubic  centimeter  of  strong  nitric  acid  to  four 
or  five  cubic  centimeters  of  white  of  egg  solution  and  warming.  Coagulation  fol- 
lows at  once.  With  a  very  dilute  albumin  solution  the  substance  separates  in 
flakes,  while  with  a  strong  solution  a  stiff,  jelly-like  mass  may  result.  The  test 
is  a  common  one  in  urine  analysis,  but  must  be  conducted  with  certain  precautions. 

Experiment.  Solutions  of  most  protein  substances  are  precipitated  by  addition 
of  alcohol  in  excess.  This  may  be  shown  by  mixing  white  of  egg  solution  with 
strong  alcohol,  the  latter  being  added  gradually  until  the  maximum  of  precipitate 
is  obtained.  With  dilute  alcohol  precipitates  are  usually  not  secured,  and  besides 
the  alcohol  precipitation  is  usually  not  a  permanent  coagulation  as  in  the  above 
case  with  the  acid. 

Experiment.  Precipitation  by  Salts.  Some  of  the  salts  of  heavy  metals  give 
characteristic  precipitates  with  protein  solutions.  The  behavior  may  be  illustrated 
by  adding  to  dilute  egg  albumin   solution   small  amounts  of  solution  of  mercuric 


56  PHYSIOLOGICAL    CHEMISTRY. 

chloride,  lead  acetate,  copper  sulphate  or  ferric  chloride.  The  reagents  must  not 
be  added  in  excess,  as  in  some  cases  this  causes  a  resolution  of  the  precipitate. 
Similar  reactions  may  be  obtained  with  solutions  of  most  of  the  heavy  metals, 
but  the  salts  mentioned  are  often  used  in  practice.  The  behavior  of  mercuric 
chloride  or  corrosive  sublimate  as  an  active  disinfecting  agent  depends  on  its 
property  of  coagulating  the  protein  matter  of  the  pathogenic  bacteria,  to  destroy 
which  it  is  used.  The  precipitates  may  be  formed  in  neutral,  acid  or  alkaline 
solution  as  a  rule,  and  chemically  may  be  regarded  as  salts  of  the  metals  used  as 
precipitants. 

Experiment.  Precipitation  by  Alkaloid  Reagents.  In  acid  solution  the  pro- 
tein bodies  are  very  generally  precipitated  by  addition  of  solutions  of  picric  acid, 
tannic  acid,  potassium-mercuric  iodide,  phosphomolybdic  acid  and  other  reagents 
employed  in  the  detection  of  alkaloids.  The  precipitates  are  voluminous,  and  in 
most  cases  complete  in  presence  of  sufficient  acid. 

White  of  egg,  much  diluted,  may  be  used  in  illustration. 

The  above  reactions  may  be  explained  on  the  assumption  that  the 
proteins  here  act  as  pseudo-acids  or  pseudo-bases.  In  perfectly  pure 
solution  they  are  neutral  and  indifferent  to  some  indicators,  but  the 
addition  of  a  mineral  acid  imparts  to  them  the  character  of  pseudo- 
ammonium  bases  which  yield  precipitates  as  the  alkaloids  do  under 
like  circumstances.  On  the  other  hand,  with  salt  solutions  they  be- 
come pseudo-acids  and  form  now  insoluble  precipitates  of  complex 
salts.  But  it  has  been  shown  that  while  some  of  the  proteins  may  be 
neutral  to  litmus  they  may  at  the  same  time  be  quite  distinctly  acid  to 
phenolphthalein,  and  require  a  decided  amount  of  decinormal  alkali 
solution  to  give  a  reaction  by  displacing  the  acid  in  combination  with 
them.  This  is  taken  to  indicate  that  they  should  be  looked  upon  as 
true  bases,  rather  than  as  pseudo-bases.  The  amount  of  acid  which 
may  unite  with  certain  proteins  has  recently  been  found  with  con- 
siderable accuracy,  and  becomes  in  some  degree  a  measure  of  the 
basicity.  In  other  cases  it  may  be  shown  that  they  have  a  true  rather 
than  a  pseudo-acid  character  and  unite  with  alkali  to  form  real  salts. 

Behavior  with  Millon's  Reagent.  In  this  we  have  one  of  the  most 
characteristic  reactions  of  the  protein  bodies.  Millon's  reagent  is  made 
by  dissolving  mercury  in  twice  its  weight  of  strong  nitric  acid,  com- 
pleting the  reaction  by  heat.  The  solution  obtained  is  diluted  with 
twice  its  volume  of  water.  It  contains  a  little  nitrous  acid.  When 
warmed  with  white  of  egg  and  other  proteins  it  imparts  a  deep  red 
color  to  the  coagulum  produced  and  often  to  the  containing  solution. 
The  reaction  is  common  to  benzene  derivatives  which  have  a  hydroxyl 
group  attached  to  the  nucleus,  and  is  given  by  phenol  for  example. 
The  reaction  in  the  protein  substances  is  due  to  the  presence  of  the 
tyrosine  group  in  the  complex  molecule.  This  group  seems  to  be 
present  in  all  protein  bodies  with  the  exception  of  gelatin,  so  that  the 


THE    PROTEIN    SUBSTANCES.  5  7 

reaction  is  a  nearly  universal  one.  The  protein  derivatives  which  still 
contain  the  tyrosine  complex  likewise  show  the  reaction.  Tyrosine  is 
represented  by  the  formula  C6H4OH.CH2.CHNH2.COOH,  and  will 
be  referred  to  later,  as  it  is  an  important  decomposition  product  of 
proteins. 

Experiment.  Test  the  behavior  of  Millon's  reagent  by  adding  some  to  white  of 
egg  solution,  milk  or  flour,  and  applying  heat.  The  characteristic  color  appears 
almost  immediately.  Its  depth  depends  somewhat  on  the  concentration  of  the 
protein  substance  used.  Presence  of  much  salt  interferes  with  the  test  or  may 
even  prevent  the  reaction. 

Experiment.  Apply  the  same  test  to  weak  solutions  of  phenol,  salicylic  acid  and 
thymol.  Note  the  color  and  character  of  the  reaction.  These  bodies  all  have  a 
benzene  nucleus  with  hydroxyl  combination.  If  pure  tyrosine  is  available  a  very 
dilute  solution  may  be  employed  for  tests.  It  is  said  that  I  part  in  iooo  of  water 
gives  a  distinct  reaction.  Hydroquinol,  resorcinol  and  a-  and  /3-naphthol  give  like- 
wise decided  reactions,  but  the  colors  are  orange  yellow. 

The  Biuret  Reaction.  This,  like  the  above,  is  a  protein  test  de- 
pending on  the  presence  of  certain  groups  in  the  complex  molecule. 
When  biuret  and  several  substances  of  related  composition  are  mixed 
with  an  excess  of  alkali  solution,  either  sodium  or  potassium  hydrox- 
ide, and  then  a  few  drops  of  a  weak  copper  sulphate  solution  are 
added,  a  blue-violet  to  reddish-violet  color  is  produced.  The  shade 
depends  on  the  concentration  of  the  solution,  and  on  the  composition 
of  the  reacting  group.  It  has  been  shown  that  the  reaction  seems  to 
follow  with  compounds  which  contain  two  groups — CONH2  directly 
united  or  joined  by  a  carbon  or  nitrogen  atom,  as  for  example : 

CONH,  yCONH,  /CONH.      CO  •  CONH2 

|  HN(  H2C<  "       I 

CONfL  XCONH2  NCONH2      NH  •  CONH2 

Oxamide  Biuret  Malondiamide  Oxaluramide 

The  combination  of  copper  and  alkali  with  these  bodies  has  been 
recently  studied  and  formulas  determined.  If,  in  place  of  using  a 
solution  of  one  of  these  compounds,  some  white  of  egg,  or  other 
protein  solution,  is  employed  the  same  color  appears.  The  absorption 
spectra  from  pure  biuret  and  egg  albumin  solution,  treated  in  this 
manner,  are  the  same,  which  shows  that  the  albumin  must  split  off  this 
group  under  the  influence  of  the  alkali  used.  The  reaction  is  one  of 
extreme  delicacy  and  may  be  employed  for  the  recognition  of  traces 
of  protein  compounds.  It  is  used  especially  in  the  detection  of  peptone, 
one  of  the  derived  protein  substances.  Derivatives  of  simpler  nature, 
that  is,  the  products  of  the  decomposition  of  proteins,  do  not  give  the 
reaction.  It  is  therefore  of  value  in  following  the  course  of  experi- 
ments on  the  digestion  or  hydrolysis  of  proteins,  as  the  reaction  dis- 
appears with  the  breaking  flown  of  the  last  protein  complex. 


58  PHYSIOLOGICAL    CHEMISTRY. 

Nickel  salts  exhibit  an  analogous  behavior,  but  show  orange  yellow 
colors.    Cobalt  solutions  give  reddish  colors,  but  not  very  strong. 

Experiment.  Prepare  a  dilute  white  of  egg  solution  and  add  to  5  cc.  of  it  some 
solution  of  potassium  or  sodium  hydroxide.  Then  add  one  or  two  drops  of  weak 
copper  sulphate  solution,  or  enough  to  impart  the  characteristic  color.  An  excess 
of  the  copper  yields  a  precipitate  and  must  be  avoided.  The  reaction  is  much 
sharper  with  albumose  and  peptone  derivatives  than  with  the  original  native  pro- 
tein. Repeat  the  test  with  solution  of  nickel  sulphate.  The  test  is  obscured  by  the 
presence  of  ammonium  salts,  which  is  sometimes  a  matter  of  importance  in  prac- 
tical work. 

The  a-Naphthol  Test  of  Molisch.  In  the  chapter  on  the  sugars 
it  was  shown  that  a  very  marked  color  reaction  is  given  by  mixing  a 
few  drops  of  a  weak  alcoholic  solution  of  a-naphthol  with  the  sugar 
solution  and  then  adding  some  strong  sulphuric  acid.  The  same  be- 
havior is  shown  by  solutions  of  some  protein  substances,  which  indi- 
cates that  they  must  contain  a  carbohydrate  group  of  some  kind.  The 
reaction  depends  on  the  formation  of  furfuraldehyde  by  the  decompo- 
sition of  the  sugar  by  the  strong  acid.  This  furfuraldehyde  combines 
then  with  the  a-naphthol  to  produce  a  deep  violet  color,  the  reaction 
being  similar  to  that  between  furfuraldehyde  and  aniline  acetate  de- 
scribed in  the  pentose  test  in  a  former  chapter. 

Experiment.  To  a  few  cubic  centimeters  of  white  of  egg  solution  add  five  drops 
of  10  per  cent,  solution  of  a-naphthol  in  alcohol.  Then  carefully  add  three  or 
four  cubic  centimeters  of  strong  sulphuric  acid,  which  sinks  below  the  lighter  solu- 
tion. Note  the  color  at  the  zone  of  contact  and  throughout  the  liquid  on  shaking. 
Alkalies  change  the  color  to  yellow.  Thymol  solution  is  sometimes  used  instead 
of  a-naphthol.  This  gives  a  deep  red  color.  These  furfuraldehyde  reactions  are 
extremely  delicate,  and  appear  in  a  great  variety  of  tests.  Their  general  character 
and  importance  should  therefore  be  recognized. 

The  Xanthoproteic  Reaction.  This  is  a  delicate  test,  depending 
on  the  formation  of  yellow  nitro  derivatives  of  the  phenol  groups 
in  the  protein  complex.  Similar  reactions  are  given  by  many  simpler 
organic  substances  where  nitric  acid  is  mixed  with  them  and  heat 
applied.  The  color  produced  by  nitric  acid  in  contact  with  the  skin 
is  due  to  the  same  general  reaction. 

Experiment.  In  illustration,  add  some  strong  nitric  acid  to  white  of  egg  solu- 
tion. On  application  of  heat  the  yellow  color  appears.  By  neutralizing  with 
ammonia  the  color  changes  to  orange  yellow. 

Make  a  similar  test  by  warming  some  phenol  solution  with  nitric  acid.  In  this 
case  a  nitro-phenol  is  formed.  Pure  phenol  and  strong  nitric  acid,  it  will  be 
recalled,  yield  trinitrophenol  or  picric  acid,  C6H2-OH-  (N02)3  Add  ammonia  to 
neutralize,  as  before. 

The  Lead  Hydroxide  Test.  The  protein  bodies  contain  sulphur 
which  may  be  removed  by  action  of  an  alkaline  lead  solution,  or 


THE    PROTEIN    SUBSTANCES.  59 

alkaline  bismuth  solution  or  mixture.  The  second  reaction  has  some 
importance,  as  it  is  the  source  of  a  fallacy  in  the  so-called  bismuth  test 
for  sugar  in  urine.  In  presence  of  albumin,  sulphide  of  bismuth  is 
formed  in  place  of  the  reduction  product  indicative  of  sugar.  As  all 
protein  bodies  contain  sulphur  the  test  is  a  general  one.  It  may  be 
made  as  follows : 

Experiment.  Produce  first  a  soluble  alkaline  compound  of  lead  by  adding  to  a 
few  cubic  centimeters  of  lead  acetate  solution  enough  strong  alkali,  sodium  or 
potassium  hydroxide,  to  form  a  precipitate  and  redissolve  it.  Then  add  the  protein 
substance,  white  of  egg  for  example,  and  boil.  A  brown  or  black  color  appears 
and  sometimes  even  a  precipitate  of  black  lead  sulphide.  Only  a  part  of  the  sul- 
phur, however,  may  be  separated  in  this  simple  manner.  Another  portion  seems 
to  be  much  more  firmly  combined  in  the  protein  molecule. 

The  reactions  which  have  just  been  explained  are  the  most  impor- 
tant and  characteristic  of  all  which  have  been  suggested  for  the  recog- 
nition and  identification  of  the  proteins.  Numerous  other  reactions 
are  known,  however,  which  are  easily  observed.  Several  of  these  are 
color  tests,  depending  on  the  formation  and  combination  of  furfur- 
aldehyde,  but  they  need  not  be  described  as  in  principle  they  do  not 
differ  much  from  the  Molisch  test. 

QUANTITATIVE    DETERMINATION    OF    PROTEINS. 

The  above  tests  serve  for  the  recognition  of  proteins  but  not  for 
their  determination,  and  for  the  latter  purpose  it  may  be  said  further 
that  no  one  method  is  perfectly  suited  to  all  cases.  Many  of  the 
simpler  protein  bodies  are  determined  by  complete  coagulation,  fol- 
lowed by  weighing  of  the  precipitate  formed.  This  involves  several 
operations,  all  of  which  must  be  very  carefully  performed.  For 
example,  a  pure  native  albumin  in  solution  may  be  coagulated  by 
adding  a  few  drops  of  acetic  acid  and  boiling  thoroughly.  The  coagu- 
lum  is  collected  on  a  weighed  paper  filter  or  in  a  Gooch  funnel,  thor- 
oughly washed,  dried  and  weighed.  Instead  of  drying  and  weighing 
the  precipitate  it  may  be  decomposed  according  to  the  Kjeldahl  process, 
in  which  the  nitrogen  is  converted  into  ammonia  by  digestion  with 
sulphuric  acid.  The  ammonia  is  easily  separated  and  measured.  The 
nitrogen  is  14/17  of  it.  By  multiplying  the  nitrogen  found  by  the 
factor  6.25  we  obtain  the  original  protein  content  on  the  assumption 
that  these  bodies  contain  16  per  cent  of  nitrogen.  This  method  is 
now  commonly  followed  in  the  determination  of  crude  protein  for 
many  technical  and  scientific  purposes.  But  in  many  cases  an  error  is 
naturally  introduced  because  of  the  uncertainty  of  the  factor;  6.25 
is  the  mean  value  for  the  native  proteins  and  the  closely  related  bodies. 


60  PHYSIOLOGICAL    CHEMISTRY. 

COMPONENT  GROUPS  IN  THE  PROTEIN  COMPLEX. 

In  the  study  of  the  protein  molecule  as  a  whole,  a  limit  is  soon 
reached  in  any  attempt  to  fix  its  composition,  but  much  may  be  learned 
by  observing  the  various  products  formed  in  reactions  by  which  the 
molecule  is  broken  down  under  the  influence  of  different  agents. 
Some  of  these  reactions  are  apparently  largely  hydrolytic  in  character, 
and  in  a  degree  may  be  compared  to  the  decomposition  of  a  fat  by 
superheated  steam.  In  this  very  simple  case  glycerol  and  fatty  acids 
are  obtained  and  we  conclude  that  they  were  not  actually  formed  in 
the  process,  but  that  they  were  present  in  combination  in  the  original 
fat.  In  treating  protein  bodies  in  a  similar  manner  or  in  subjecting 
them  to  the  decomposing  influences  of  acids  or  alkalies,  a  number  of 
products  are  formed.  These  must  be  either  results  of  peculiar  disinte- 
gration and  subsequent  synthesis,  or  they  must  represent  groups  in 
some  way  existent  in  the  original  complex.  The  latter  view  is 
strengthened  by  the  fact  long  observed  that  certain  products  result, 
whatever  the  method  of  decomposition.  Leucine,  for  example,  is 
found  abundantly  among  the  products  liberated  by  subjecting  protein 
to  the  action  of  superheated  steam,  hot  hydrochloric,  nitric  or  dilute 
sulphuric  acid,  concentrated  alkali  solutions,  bromine  water  under 
pressure,  or  to  prolonged  pancreatic  digestion.  The  almost  necessary 
conclusion  must  be  that  in  these  varied  reactions  the  leucine  could  not 
have  formed  from  smaller  disintegration  groups,  but  must  have  been 
set  free  from  something  holding  it  in  the  protein  complex. 

The  decomposition  reactions  are  therefore  considered  very  impor- 
tant as  suggesting  probably  the  component  groups  in  the  large  mole- 
cule. In  the  following  pages  a  few  of  the  most  important  of  these 
reactions  and  their  products  will  be  described. 

Decomposition  by  Steam  under  Pressure.  By  prolonged  heating 
of  protein  substances  with  water  certain  changes  take  place,  even 
below  a  temperature  of  ioo°  C.  Following  the  coagulation,  which 
appears  in  most  cases,  a  gradual  hydration  and  solution  begins,  and  a 
small  portion  of  the  substance  is  brought  into  the  form  of  albumose  or 
possibly  peptone.  At  a  higher  temperature,  that  is,  by  heating  with 
water  or  steam  under  pressure,  more  profound  changes  take  place. 
Ammonia  and  hydrogen  sulphide  are  split  off  from  the  molecule  and 
relatively  large  amounts  of  albumose  and  peptone  are  formed.  If  the 
temperature  is  high  enough  the  reaction  extends  to  the  complete  de- 
struction of  the  molecule  and  such  bodies  as  leucine  and  tyrosine  are 
produced  in  quantity. 


THE    PROTEIN    SUBSTANCES.  6 1 

Effect  of  Alkalies.  Much  more  decided  changes  are  noticed  when 
the  protein  body  is  heated  with  alkali  solutions.  Experiments  of  this 
kind  were  long  ago  carried  out  by  Schiitzenberger  and  have  since  been 
frequently  repeated.  Numerous  compounds  have  been  identified 
among  the  decomposition  products,  and  of  these  the  most  important 
are  leucine  in  quantity,  tyrosine,  ammonia,  carbonic  acid,  butyric 
acid,  formic  acid,  acetic  acid,  oxalic  acid,  aspartic  acid,  amino-valeric 
and  amino-butyric  acid.  Barium,  potassium  and  sodium  hydroxide 
solutions  have  been  used  for  the  purpose.  By  melting  the  dry  pro- 
teins with  alkali  some  of  the  same  products  are  formed,  especially 
leucine  and  tyrosine. 

Effect  of  Acids.  Many  experiments  have  been  made  on  the  de- 
composition of  protein  bodies  by  boiling  with  acids,  and  particularly 
with  strong  hydrochloric  acid,  the  hydrolyzing  power  of  which  is 
very  great.  The  most  important  of  the  products  isolated  in  this  way 
are  the  following: 

The  Hexone  Bases.  The  term  hexone  bases  has  been  given  by 
Kossel  to  a  group  of  bodies  which  occur  commonly  in  the  decomposi- 
tion products  of  practically  all  the  protein  substances.  We  have  here 
arginine,  C6H14N402,  lysine,  C6H14N202,  and  histidine,  C6H9N302. 
The  first  appears  to  be  a  guanidine  derivative  of  amino-valeric  acid, 
the  second  is  diamino-caproic  acid,  while  the  third  appears  to  be  a 
diamine  acid  of  composition  as  yet  unknown,  or  is,  possibly,  an  imino- 
azol  derivative  of  amino-propionic  acid.  The  isolation  of  these  com- 
pounds was  a  very  important  step  in  the  direction  of  clearing  up  the 
constitution  of  the  proteins,  inasmuch  as  some  of  the  simplest  of  these 
bodies,  the  protamines,  seem  to  consist  almost  wholly  of  the  hexones. 
More  will  be  said  about  this  relation  later.  The  hexones  are  soluble, 
crystalline,  optically  active,  compounds,  and  because  of  their  wide 
occurrence  have  been  very  thoroughly  studied.  They  contain  the 
amino  group  in  the  a  position,  and  in  this  respect  resemble  the  other 
common  disintegration  products.  All  the  a  amino  acids  appear  to 
have  a  sweetish  taste,  which  is  illustrated  by  the  first  of  the  products 
to  follow. 

Glycocoll  or  Glycine,  C2H5N02,  amino-acetic  acid.  Obtained 
abundantly  from  gelatin  and  also  from  a  few  other  proteins.  It  is 
very  soluble  in  water  to  which  it  imparts  a  sweetish  taste,  and  is 
insoluble  in  alcohol  or  ether.  From  a  theoretical  standpoint  the  im- 
portance of  glycine  is  very  great,  as  it  is  the  starting  point  in  various 
syntheses,  to  be  explained  later.     It  also  combines  with  benzoic  acid 


62 


PHYSIOLOGICAL    CHEMISTRY. 


to  form  hippuric  acid  in  the  body  metabolism.  Hippuric  acid  is 
benzoyl  glycine. 

Amino  Propionic  Acid,  or  Alanine,  C3H7N02.  This  is  in  a 
sense  the  nucleus  substance  corresponding  to  tyrosine  and  phenyl- 
alanine, to  be  referred  to.  It  is  a  soluble  product  rather  widely  dis- 
tributed in  protein  bodies,  and  because  of  this  marked  solubility  is 
hard  to  isolate. 

Amino  Valeric  Acid,  C5H11N02.  This  substance  is  apparently 
the  a  product.  It  is  usually  mixed  with  leucine  and  is  separated  only 
with  difficulty  from  this  body.  Combined  with  guanidine, 
NHC(NH2)2,  it  yields  the  important  arginine,  referred  to  above. 
Diamino  valeric  acid  is  known  as  ornithine. 

Leucine,  C6H13N02,  a-amino-caproic  acid,  or  a-amino-isobutyl- 
acetic.  acid.  This  has  been  already  mentioned  as  found  abundantly 
among  the  products  of  protein  decomposition  by  other  agencies.  In 
some  cases  it  appears  to  constitute  30  per  cent  of  the  reaction  products, 
and  must  therefore  play  a  very  important  part  in  the  original  complex 
molecule.  Leucine  is  found  in  several  different  forms;  the  common 
product  obtained  by  acid  hydrolysis  is  right  rotating  and  shows  in 
hydrochloric  acid  solution  [a]D  =  -f-  18. 90.  In  water  solution  it 
rotates  in  the  opposite  direction. 

Serine,  C3H7NOs,  amino-hydroxy-propionic  acid,  was  first  discov- 
ered in  silk,  and  hence  the  name.  But  it  is  present  in  many  of  the 
protein  bodies  as  well,  although  in  not  very  large  amount. 

Aspartic  Acid,  C4H7N04,  amino-succinic  acid.  Slightly  soluble 
in  water,  the  solutions  being  apparently  left  rotating  at  the  ordinary 
temperature.  But  in  hydrochloric  acid  it  is  strongly  right  rotating. 
While  the  acid  is  found  commonly  in  proteins  the  amounts  are  not 
large. 

Glutaminic  Acid,  C5H9N04,  a-aminoglutaric  acid.  The  acid  is 
found  in  several  optical  modifications.  The  common  form  is  but 
slightly  soluble  in  water  and  is  right  rotating.  In  hydrochloric  acid 
the  right  rotation  is  marked.  This  acid  is  obtainable  in  considerable 
quantities  from  many  proteins,  and  is  one  of  the  extremely  important 
constituent  groups.     It  is  probably  more  abundant  than  leucine. 

Proline,  C5H9N02,  (C4H7.NHCOOH),  a-pyrrolidine  carboxylic 
acid.  From  the  conditions  under  which  it  has  been  found  this  inter- 
esting body  is  supposed  to  be  a  primary  product.  It  is  an  imino  not  an 
amino  derivative.  It  was  first  obtained  from  casein  and  later  from 
other  proteins.    It  has  a  very  sweet  taste  and  a  high  left  rotation.    The 


THE    PROTEIN    SUBSTANCES.  63 

closely  related  hydroxy-a-pyrrolidine  carboxylic  acid  has  been  obtained 
from  gelatin. 

Tyrosine,  CgHuNOs,  />-oxyphenyl  amino-propionic  acid.  This 
appears  to  be  a  component  part  of  all  the  common  proteins,  with  the 
exception  of  gelatin,  and  from  some  of  them  has  been  obtained  in 
quantity.  As  already  mentioned  this  is  probably  the  substance  which 
reacts  commonly  with  Millon's  reagent.  Tyrosine  is  but  slightly 
soluble  in  water,  from  which  it  crystallizes  in  bundles  of  fine  needles. 
Its  solutions  are  optically  active.  In  presence  of  hydrochloric  acid 
[a]D= — 8°  to  —  150,  the  rotation  varying  with  the  amount  of  acid. 

Phenylalanine,  CgHuNOg,  phenylamino  propionic  acid.  This 
substance  resembles  tyrosine  closely  in  structure  and  behavior  and  is 
a  common  product  of  protein  decomposition.  It  is  slightly  soluble 
in  water  and  has  a  sweetish  taste.  It  appears  to  be  present  in  many 
cases  when  tyrosine  is  lacking,  and  must  be  considered  as  a  very 
important  decomposition  product. 

Tryptophane,  CnH12N202,  indol-amino-propionic  acid.  This 
complex  product  has  been  obtained  from  several  of  the  proteins,  and 
probably  occurs  in  most  of  them.  Many  of  the  peculiar  color  reactions 
of  proteins  are  due  to  the  small  amount  of  tryptophane  present.  The 
most  characteristic  of  these  color  reactions  is  given  by  the  addition  of 
bromine  water  to  a  liquid  containing  the  substance.  A  marked  violet 
color  results.  This  is  shown  well  in  advanced  stages  of  the  tryptic 
digestion  of  proteins.    More  will  be  said  about  the  body  later. 

From  the  above  list  of  decomposition  products  it  will  be  seen  that 
the  comparatively  simple  amino  acids  predominate;  most  of  them, 
possibly  all,  are  the  a  compounds,  and  as  will  be  shown  below,  they 
make  up  a  very  large  portion  of  the  whole  protein  molecule. 

Glucosamine,  C6Hn05(NH2).  This  appears  to  be  an  important 
constituent  in  some  groups  of  protein  bodies.  It  has  been  obtained  in 
quantity  from  the  glucoproteids  and  is  possibly  present  in  small 
amount  in  all.  It  is  usually  obtained  as  a  salt,  hydrochloride  or  hydro- 
bromide,  which  is  readily  soluble  in  water  and  optically  active.  It  is 
regarded,  usually,  as  a  secondary  product  of  dissociation. 

Ammonia,  XH,.  This  is  always  found  in  relatively  large  amount, 
but  in  the  main  may  be  a  secondary  product. 

Sulphur  Compounds.  Hydrogen  sulphide,  ethyl  sulphide,  thio- 
lactic  acid,  C3H6S02,  cystin,  C0Hi2N2S2O4,  and  traces  of  other  bodies 
which  contain  sulphur  have  been  identified  in  small  amount  among 
the  decomposition  products.     Of  these  sulphur  compounds  cystin  is 


64  PHYSIOLOGICAL    CHEMISTRY. 

the  most  important.  It  exists  in  two  isomeric  forms,  one  of  which  is 
found  in  certain  calculi. 

Carbonic  Acid  is  apparently  a  constant  derivative,  but  may  appear 
as  a  result  of  some  secondary  reaction.  Its  significance,  therefore,  is 
obscure. 

With  acids  other  than  hydrochloric  very  similar  reaction  products 
are  secured.  It  will  be  shown  below  that  the  effects  of  prolonged 
tryptic  digestion  are  very  nearly  the  same  as  observed  with  hydro- 
chloric acid.  This  is  a  point  of  the  highest  practical  importance,  as  it 
gives  us  some  insight  into  the  complex  physiological  process.  And 
in  peptic  digestion  also,  where  very  weak  hydrochloric  acid  and  pep- 
sin are  employed,  essentially  the  same  products  result  provided  the 
time  of  the  action  be  made  sufficiently  long. 

RESULTANT    CHARACTER    OF    THE    PROTEIN    MOLECULE. 

While  the  various  decompositions  detailed  above  give  us  some  in- 
sight into  the  number  and  kind  of  groups  combined  in  the  protein  com- 
plex, they  do  not,  unfortunately,  show  us  much  as  to  the  manner  in 
which  these  groups  are  combined.  We  are  not,  as  yet,  able  to  picture 
to  ourselves  a  large  molecule  in  which  the  leucine,  tyrosine,  aspartic 
acid,  glycocoll,  and  so  on,  are  united  to  form  a  molecule  with  the 
general  properties  and  molecular  weight  as  large  as  we  assign  to  even 
the  simplest  proteins,  but  a  step  has  been  made  in  that  direction 
through  the  synthesis  of  various  polypeptides  carried  out  by  Fischer 
and  Curtius,  who  have  succeeded  in  condensing  several  amino  acids 
into  one  molecule  with  certain  properties  suggesting  those  of  the 
peptones.  These  bodies  will  be  referred  to  in  a  later  chapter.  Hof- 
meister  has  suggested  the  possibility  of  the  combination  of  amino 
acids  in  large  groups  by  the  following  general  scheme : 

—  NHCHCO  —  NHCHCO  —  NHCHCO  —  NHCHCO  — 

I  I  I  I 

CH2       CH2       CH2      (CH2)3 

CH3-CH-CH3  C,HtOH         COOH  CH2NH2 

Leucine  Tyrosine  Aspartic  Acid  Lysine 

The  recognition  of  the  various  component  groups  suggests  some 
reasons  why  the  proteins  may  exhibit  acid  and  basic  behavior  at  the 
same  time.  Of  most  of  these  protein  compounds  the  basic  character 
is  the  more  pronounced  and  more  readily  observed ;  that  is,  their  acid 
combining  power.  Some  writers  consider  these  bodies  as  so-called 
pseud o  bases  and  pseudo  acids,  because  of  the  very  peculiar  manner 
in  which  they  unite  with  acids  and  bases.     But  several  investigations 


THE    PROTEIN    SUBSTANCES.  6$ 

of  the  last  few  years  indicate  that  they  are  more  properly  true  bases 
and  acids,  but  so  weak  in  their  combinations  that  hydrolysis  follows 
very  readily.  This  hydrolysis  obscures  the  reactions  which  must  take 
place  in  the  formation  of  salts.  In  aqueous  solutions  the  pure  proteins 
and  the  component  amino  acids  are  practically  non-electrolytes,  which 
has  been  explained  on  the  assumption  that  the  basic  part  of  one  group 
is  linked  to  the  acid  part  of  another,  with  little  or  no  dissociation. 
Possibly,  also,  a  ring-like  structure  is  formed  by  a  kind  of  internal 
saturation.  By  various  methods  it  may  be  shown  that  the  simple  pro- 
teins and  the  amino  acids  combine  in  rather  definite  proportions  with 
the  inorganic  acids  and  bases,  as  will  be  pointed  out  in  later  chapters. 
But  the  salts  so  formed  suffer  marked  hydrolytic  dissociation  and 
conduct  the  electric  current  essentially  as  the  acid  or  base  used.  Gly- 
cocoll  hydrochloride,  for  example,  CH2NH2.HC1 — COOH,  hydro- 
lyzes  so  as  to  leave  free  hydrochloric  acid,  while  sodium  glycocollate, 
CH2NH2 — COONa,  hydrolyzes  to  yield  sodium  hydroxide,  and  the 
conductivity  observed  is  due  to  this  latter  essentially.  With  casein, 
which  is  a  protein  easily  obtainable  in  pure  condition  from  milk,  the 
phenomena  of  salt  formation  both  with  acids  and  bases  may  be  very 
easily  observed.  Something  will  be  said  about  this  later.  One  mole- 
cule of  casein  combines,  apparently,  with  four  or  five  molecules  of 
sodium  hydroxide  to  form  a  salt.  In  their  basic  capacity  serum  and 
egg  albumins  combine  with  a  large  number  of  molecules  of  hydro- 
chloric acid. 

The  presence  of  sulphur  in  the  proteins  was  shown  by  a  test  referred  to  some 
pages  back.  Investigations  have  shown  that  sulphur  is  present  in  at  least  two  kinds 
of  combinations  in  the  protein  complex;  there  must  be  at  least  two  sulphur  atoms 
in  the  molecule.  Some  of  the  sulphur  is  easily  separated  by  hot  alkali  solutions, 
while  the  rest  of  it  is  not.  No  part  of  this  element  appears  to  be  combined  in  oxi- 
dized form,  that  is,  in  the  condition  of  a  sulphite  or  sulphate.  The  sulphur  com- 
pounds which  have  been  obtained  in  protein  decomposition  are  such  as  may  be 
derived  from  a  breaking  down  of  the  cystin  group.  It  has  been  shown  that  cystin 
gives  up  its  sulphur  very  slowly  to  boiling  alkali,  and  only  in  part  as  sulphide. 

The  general  reactions  and  characteristics  of  the  protein  bodies  hav- 
ing been  discussed,  a  brief  description  of  the  more  important  indi- 
vidual substances  will  now  follow. 

TRUE    OR    NATIVE    ALBUMINS. 

In  the  scheme  of  classification  given  some  pages  back  the  true  or 
native  albumins  have  the  first  place.     The  best  known  representatives 
of  the  protein  group  are  included  here. 
6 


66  PHYSIOLOGICAL    CHEMISTRY. 

ALBUMINS    PROPER. 

These  bodies  are  characterized  by  solubility  in  water  and  in  weak 
cold  acid  or  alkali  solutions.  They  are  readily  coagulated  by  heat 
and  by  shaking  with  strong  alcohol.  Although  usually  considered  as 
amorphous,  the  albumins  have  been  obtained  in  well  crystallized  form, 

The  characteristic  color  reactions  previously  referred  to  are  all 
given  by  the  true  albumins  and  they  are  precipitated  by  ammonium 
sulphate  or  zinc  sulphate  added  to  saturation.  With  strong  sodium 
chloride  precipitation  follows  only  after  addition  of  acid. 

Serum  Albumin.  This  is  the  important  protein  body  of  blood 
serum,  of  which  it  constitutes  three  to  four  per  cent  by  weight,  the 
related  substance,  serum  globulin,  making  up  nearly  as  much.  While 
closely  resembling  each  other,  it  is  not  definitely  known  that  the  serum 
albumins  of  different  animals  are  identical.  In  fact,  certain  reactions 
to  be  referred  to  later  suggest  peculiar  points  of  difference.  In  blood, 
the  albumins  are  associated  with  globulins,  fibrinogen,  mucoids,  salts 
and  other  bodies,  the  perfect  separation  of  which  is  practically  im- 
possible. The  purification  of  serum  albumin  by  crystallization  is  not 
easily  carried  out  with  all  blood  serums;  in  some  cases  the  formation 
of  crystals  is  slow  and  incomplete. 

Serum  albumin  contains  a  relatively  large  amount  of  sulphur,  about 
two  per  cent  in  the  mean,  and  is  characterized  further  by  a  high 
specific  rotation.  The  values  which  have  been  given  for  this  are  not 
constant,  but  in  the  mean  are  about  [a]D= — 6o°. 

Crude  serum  albumin  is  now  an  article  of  commerce,  being  made  in 
large  quantities  from  blood  collected  at  the  slaughtering  houses.  It 
is  usually  mixed  with  globulin,  and  besides  is  partly  insoluble  because 
of  the  high  temperature  employed  in  drying  it.  For  the  following 
experiments  fresh  blood  must  be  used. 

Experiment.  Collect  blood  in  a  clean  vessel  and  stir  it  thoroughly  to  separate 
the  fibrin  and  part  of  the  corpuscles,  as  a  clot.  Some  of  the  corpuscles,  however, 
remain  with  the  serum  and  may  be  separated  by  allowing  the  latter  to  stand  in  a 
tall,  narrow  jar,  or  better,  by  rotating  the  serum  in  a  centrifugal  machine.  Most 
of  the  corpuscles  may  be  deposited  in  this  way,  leaving  a  yellowish  liquid.  A  pure 
white  serum  can  not  be  obtained  because  a  little  of  the  hemoglobin  dissolves  from 
the  corpuscles  and  remains  in  solution.  With  this  prepared  serum  make  the  fol- 
lowing tests : 

Experiment.  To  a  little  of  the  serum  add  finely  powdered  magnesium  sulphate 
to  saturation ;  this  produces  a  precipitation  of  serum  globulin,  which  separates  on 
standing.  Pour  off  the  clear  liquid  and  add  to  it  powdered  ammonium  sulphate, 
which  gives  now  a  precipitate  of  albumin. 

Experiment.  Mix  a  little  of  the  serum  with  two  or  three  volumes  of  water  in 
a  test-tube,  and  test  the  temperature  of  coagulation.     It  will  be  found  near  jo°  C. 


THE    PROTEIN    SUBSTANCES. 


67 


Lactalbumin.  Milk  contains  two  protein  substances,  the  most 
important  of  which  is  casein.  The  other  is  a  true  albumin  which  is 
present  to  the  extent  of  about  one-half  per  cent  in  cow's  milk.  It 
resembles  serum  albumin  very  closely  but  appears  to  have  a  much 
lower  specific  rotation,  [a]D  —  —  380. 

Egg  Albumin.  White  of  egg  contains  this  body  as  its  character- 
istic constituent  along  with  some  globulin  and  mucoid,  and  traces  of 
salts.  Common  albumin  reactions  are  usually  made  with  white  of  egg 
solution.  Although  this  substance  is 
always  described  as  a  true  albumin, 
some  of  its  reactions  seem  to  suggest 
that  it  may  belong  to  the  group  of 
glucoproteids,  or,  at  any  rate,  may 
contain  such  a  compound  in  rela- 
tively large  amount.  On  heating  egg 
albumin  with  weak  acid  glucosamine 
is  split  off  and  in  quantity  sufficient 
to  indicate  a  rather  large  sugar  con- 
tent in  the  original  substance.  The 
specific  rotation  is  much  lower  than 
that  of  serum  albumin  and  may  be 
taken  at  [a]D  =  — 380,  as  for  milk 
albumin.  Besides  this  difference,  egg 
albumin  has  a  much  lower  coagulat- 
ing temperature  than  has  been  given 
for  serum  albumin,  viz.,  560.  Egg 
albumin  is  much  more  easily  coagu- 
lated by  ether  than  is  serum  albumin. 
Egg  albumin  becomes  very  quickly 
insoluble  when  mixed  with  strong 
alcohol.  From  serum  albumin  it  differs,  further,  by  this  interesting 
property.  When  its  solution  is  injected  into  the  blood  circulation  it 
passes  unchanged  through  the  kidneys  into  the  urine ;  the  same  thing 
happens  when  large  quantities  of  white  of  egg  are  eaten.  It  seems  to 
escape  digestion  in  this  latter  case  and  be  absorbed  in  pure  condition,  to 
be  later  discarded  by  the  kidneys.  These  various  points  of  behavior 
indicate,  then,  a  rather  marked  difference  between  the  two  kinds  of 
albumin. 

Experiment.  So-called  pure  egg  albumin  may  be  obtained  in  this  way :  The 
white  of  egg  is  shaken  in  a  bottle  with  some  broken  glass  to  thoroughly  break  up 
the  membranes.     The  foamy  mass  js  filtered  through  fine,  unsized  muslin,  and  to 


Fig.  8.  Typical  form  of  Graham 
dialyzer  frequently  used  in  purifica- 
tion of  proteins.  The  substance  to  be 
purified  is  placed  in  the  cell  a,  which 
has  a  parchment  bottom  and  floats  on 
water  in  the  large  vessel  b.  The  simple 
parchment  tube  dialyzers  now  obtain- 
able  are    more   efficient. 


68  PHYSIOLOGICAL    CHEMISTRY. 

the  filtrate  an  equal  volume  of  saturated  ammonium  sulphate  solution  is  added. 
This  produces  a  precipitate  of  globulin  which  after  24  hours  is  filtered  off.  Am- 
monium sulphate  in  this  strength  does  not  precipitate  the  true  albumin.  To  this 
filtrate  a  little  more  saturated  ammonium  sulphate  is  added  and  until  a  precipitate 
or  turbidity  just  begins  to  show.  This  is  caused  to  disappear  by  the  cautious 
addition  of  water,  a  few  drops  at  a  time.  Finally,  acetic  acid  of  ten  per  cent 
strength  saturated  with  ammonium  sulphate  is  added  until  a  turbidity  again  appears, 
and  then  the  mixture  is  allowed  to  stand  24  hours  in  a  cool  place.  A  part  of  the 
albumin  separates  in  the  crystalline  form.  This  is  collected,  redissolved  in  a  very 
little  cold  water  and  reprecipitated  with  ammonium  sulphate  and  acetic  acid  as 
before.  The  crystals  are  collected  on  a  filter,  then  transferred  to  a  dialyzer  with 
water  for  the  separation  of  the  sulphate  by  dialysis.  In  this  way  a  nearly  pure 
albumin  may  be  obtained  in  solution,  but  the  crystallized  substance  has  not  been 
secured  free  from  salts. 

White  of  egg  contains  in  the  mean  about  86  per  cent  of  water,  13 
per  cent  of  proteins,  0.6  per  cent  of  mineral  matters  and  a  little  fat. 
The  yellow  of  egg  is  a  substance  of  very  different  composition.  The 
water  present  amounts  to  about  50  per  cent,  the  proteins  to  16  per 
cent,  the  fat  to  30  per  cent,  or  more,  while  the  ash  is  about  1  per  cent. 
The  fat  contains  a  notable  quantity  of  lecithin. 

GLOBULINS. 

The  proteins  of  this  group  differ  from  the  albumins  mainly  with 
respect  to  solubility  in  water.  In  pure  water  they  are  practically 
insoluble,  but  they  dissolve  in  moderately  dilute  salt  solutions.  On 
diluting  a  globulin  solution  of  this  kind  precipitation  follows.  Globu- 
lin solutions  coagulate  by  heat  in  much  the  same  manner  as  observed 
with  albumins,  but  in  general  they  become  permanently  insoluble 
even  more  readily  than  do  the  albumins. 

The  preparation  of  pure  globulin  is  even  more  difficult  than  the 
preparation  of  pure  albumin.  The  globulin  must  first  be  separated 
by  precipitation  with  some  salt ;  as  the  salt  is  later  removed  by  dialysis 
the  globulin  remaining  becomes  insoluble,  which  makes  further  treat- 
ment difficult.     Globulins  are  not  well  known  in  crystalline  condition. 

Serum  Globulin.  This  substance  makes  up  a  large  fraction  of 
the  protein  in  blood  serum,  amounting  to  nearly  as  much  as  the  serum 
albumin.  For  a  long  time  it  was  confounded  with  the  latter,  and  it 
was  only  after  a  lengthy  series  of  investigations  by  different  chemists 
that  its  true  nature  was  recognized.  This  globulin  may  be  discovered 
easily  in  the  serum  when  the  latter  body  is  diluted  with  water,  but  the 
separation  is  never  quite  complete  by  the  water  treatment  alone,  as  a 
portion  always  remains  in  solution.  By  salting  out  with  ammonium 
sulphate  to  half  saturation,  or  with  magnesium  sulphate  completely, 
the  desired  end  is  reached. 


THE    PROTEIN    SUBSTANCES.  69 

The  coagulation  temperature  of  serum  globulin  is  given  as  75  °  and 
the  specific  rotation  as  [a]fl  = —  4§°>  but  these  numbers  are  some- 
what uncertain,  especially  the  latter. 

Bence-Jones  Proteid.  This  is  the  name  given  to  a  substance  oc- 
casionally found  in  pathological  urines,  and  which  has  usually  been 
considered  an  albumose.  When  purified,  however,  it  has  been  found 
to  have  the  properties  of  a  globulin.  It  may  be  held  in  solution  in  the 
urine  by  the  salts  present. 

Other  Globulins.  Several  other  bodies  are  described  as  globulins. 
The  most  important  of  these  is  the  so-called  cell  globulin,  which  is 
possibly  identical  with  serum  globulin.  This  substance  has  been  ob- 
tained from  different  organs,  from  the  liver,  from  the  pancreas,  from 
muscle  plasma,  etc.  Some  of  the  globulins  described  as  cell  globulins 
have  a  lower  coagulating  temperature  than  the  true  serum  globulin. 

In  the  crystalline  lens  of  the  eye  a  body  has  been  long  known  which 
is  called  crystallin.  In  coagulation  temperature  and  specific  rotation 
this  crystallin  appears  distinct  from  serum  globulin,  and  further,  it 
seems  to  be  made  up  of  two  related  substances,  a  and  /?  crystallins. 

Globulins  have  been  described  in  milk  and  egg  and  also  in  the 
vegetable  kingdom  under  the  name  of  phy 'to globulins  or  phytovitellins. 
This  last  designation  indicates  that'  they  may  be  classed  under  the 
head  of  the  nucleo-albumins,  with  which  bodies  they  have  much  in 
common.  Among  the  best  known  of  these  bodies  we  have  the  abun- 
dant protein  called  edestin. 

Edestin.  According  to  Osborne  this  is  a  true  globulin,  and  of 
the  vegetable  products  of  this  class  has  been  among  the  most  thor- 
oughly studied.  It  has  been  obtained  from  many  seeds  and  nuts,  but 
most  readily  from  hemp  seed.  On  analysis  it  shows,  in  the  mean, 
about  18.7  per  cent  of  nitrogen,  and  0.9  per  cent  of  sulphur.  Its 
specific  rotation  is  about  — 440.  Edestin  can  be  secured  in  the  crys- 
talline condition,  which  has  facilitated  greatly  its  study.  When  hemp 
seed  meal  is  extracted  with  sodium  chloride  solution  and  this  is  fol- 
lowed by  dialysis  or  sharp  cooling  a  portion  of  the  edestin  separates 
in  the  crystalline  form.  The  name  edestan  is  given  to  a  slightly  hy- 
drolyzed  form  of  the  original  substance.  The  ending  an  is  employed 
in  describing  primary  protein  derivatives,  formed  by  the  action  of 
water  or  weak  acids.  These  protcans  are  insoluble  in  salt  solution,  as 
well  as  in  water. 

COAGULATING   PROTEINS. 

Several  extremely  important  substances  belong  in  this  group,  which, 
like   fibrinogen,  have  the  property  of   spontaneous   coagulation.      In 


70  PHYSIOLOGICAL    CHEMISTRY. 

nature  they  exist  normally  in  the  soluble  and  dissolved  form,  from 
which,  under  certain  influences,  not  always  well  understood,  they  pass 
to  the  solidified  condition.  This  coagulation  is  a  different  thing  from 
that  produced  by  heating  to  a  high  temperature  or  by  the  addition 
of  reagents ;  the  changes  in  the  latter  case  seem  to  be  more  profound. 
We  use  in  English  the  term  coagulation  to  describe  both  classes  of 
alterations,  which  are  really  of  a  very  different  character,  as  will 
appear  from  what  follows. 

Fibrinogen.  Blood  contains  a  peculiar  protein  body  in  small 
amount,  to  which  it  owes  its  property  of  spontaneous  coagulation. 
This  body  is  called  fibrinogen  and  the  product  of  coagulation  is  known 
as  fibrin.  The  nature  of  and  important  factors  in  this  change  have 
been  long  subjects  of  investigation  and  discussion;  it  can  not  be  said 
that  the  matter  has  been  fully  explained  in  all  its  bearings.  The  essen- 
tial points  of  what  is  known  will  be  given  in  the  chapter  on  the  blood. 

As  a  chemical  substance  fibrinogen  is  not  known  in  perfectly  pure 
condition,  since  to  hold  it  in  soluble  form  various  agents  must  be 
added  to  the  blood.  But  the  fibrin  formed,  doubtless  through  ferment 
action,  is  easily  obtained  and  its  properties  are  well  established.  As 
usually  prepared  it  is  a  white,  elastic,  stringy  mass,  insoluble  in  water, 
but  somewhat  soluble  in  salt  solutions.  Like  other  proteins  it  under- 
goes true  coagulation  through  elevation  of  temperature  or  action  of 
various  reagents.  Fibrinogen,  as  prepared  by  salting  out  from  plasma 
at  a  low  temperature,  coagulates  when  warmed  to  560.  Its  specific 
rotation  has  been  found  only  in  presence  of  salt  or  alkali  and  varies 
from  [a]I)  =  —  360  to  — 53 °  according  to  the  nature  of  the  admix- 
ture or  method  of  preparation.  It  undergoes  digestion  with  the  body 
ferments  very  readily  and  has  therefore  often  been  used  as  a  starting 
point  in  digestion  experiments. 

Myosin  and  Myogen.  The  living  muscle  plasma  contains  a  num- 
ber of  protein  substances,  one  of  which,  at  least,  possesses  the  prop- 
erty of  spontaneous  coagulation  as  observed  in  the  solidification  of  the 
muscle  after  death.  At  one  time  the  term  myosin  was  applied  to  this 
body  and  it  was  supposed  to  be  very  simple  in  nature.  Numerous 
investigations,  however,  have  shown  that  the  chemistry  of  the  muscle 
proteins  is  comparatively  complex  and  that  the  results  of  experiments 
do  not  well  agree.  In  the  older  sense  this  myosin  was  assumed  to  be 
derived  from  a  preexisting  body,  myosinogen,  in  the  living  muscle, 
much  as  fibrin  is  considered  as  derived  from  fibrinogen.  The  solidi- 
fied myosin  behaves  as  a  globulin,  which  may  be  illustrated  by  the 
following  experiment : 


THE    PROTEIN    SUBSTANCES.  7 1 

Experiment.  Free  muscle  (round  steak)  as  far  as  possible  from  traces  of  fat 
and  sinews,  and  then  thoroughly  disintegrate  it  by  passing  through  a  sausage  mill. 
Then  wash  it  repeatedly  with  cold  water  until  the  latter  is  no  longer  reddened,  and 
the  residue  appears  white.  This  is  placed  in  a  ten  per  cent  solution  of  ammonium 
chloride  and  allowed  to  remain  about  a  day,  with  occasional  shaking.  Myosin 
dissolves  in  the  ammonium  chloride  and  is  found  in  the  filtrate  when  the  mixture 
is  filtered.  Pour  the  filtrate  into  twenty  times  its  volume  of  distilled  water,  which 
causes  a  precipitation  of  the  insoluble  myosin.  Allow  to  settle  and  wash  three 
times  by  decantation.  Collect  the  precipitate  and  observe  that  portions  of  it  dis- 
solve readily  in  ten  per  cent  solutions  of  sodium  chloride  and  ammonium  chloride, 
or  in  a  o.i  per  cent  solution  of  hydrochloric  acid.  The  solution  in  salt  is  pre- 
cipitated by  the  addition  of  more  to  saturation. 

By  this  treatment  with  the  dilute  ammonium  chloride  solution  nearly 
all  of  the  protein  of  the  muscle  plasma  may  be  removed,  leaving  the 
stroma.  It  is  now  pretty  generally  recognized  that  this  solution  con- 
tains two  substances  instead  of  one.  The  first  of  these  is  still  called 
myosin,  and  is  said  to  make  up  about  20  per  cent  of  the  plasma  protein, 
while  the  name  myogen  is  given  to  the  other,  constituting  80  per  cent 
of  the  soluble  protein.  Myosin  is  the  part  of  the  plasma  which  co- 
agulates or  solidifies  the  most  readily  and  may  be  separated  from  the 
plasma  by  adding  ammonium  sulphate  to  make  28  per  cent  of  the 
solution.  On  filtering,  the  myogen  may  be  separated  by  adding  am- 
monium sulphate  nearly  to  complete  saturation.  The  coagulation 
temperature  of  myosin  is  given  as  470,  while  that  of  myogen  is  560. 
The  former  becomes  quickly  insoluble  on  addition  of  alcohol,  while 
myogen  seems  to  be  partly  soluble  in  alcohol.  Myosin-fibrin  and 
myogen-fibrin  are  the  names  given  to  the  coagulated  forms  of  these 
bodies.  More  will  be  said  of  these  relations  when  we  come  to  con- 
sider the  muscular  substance  as  a  whole. 

NUCLEO-ALBUMINS. 

This  group  contains  bodies  which  in  the  pure  state  are  rather 
markedly  acid  in  character.  They  are  called  nucleo-albumins  because 
of  the  earlier  fancied  resemblance  to  the  nucleo-proteids.  The  char- 
acteristics of  the  latter  group,  such  as  the  presence  of  nucleic  acid  and 
the  xanthine  bases  among  the  decomposition  products,  are  wholly 
wanting  in  the  nucleo-albumins.  Both  groups  contain  phosphorus, 
and  in  both  cases  the  phosphorus  is  separated  in  complex  combinations 
on  digestiorl  with  pepsin  and  hydrochloric  acid;  the  character  of  the 
phosphorus  compound  separated  is  very  different  in  the  one  case, 
however,  from  what  it  is  in  the  other. 

The  free  acids  are  but  slightly  soluble  in  water,  but  in  the  salt  form 
they  are  very  soluble  and  these  solutions  do  not  coagulate  on  boiling, 


72  PHYSIOLOGICAL    CHEMISTRY. 

as  shown  by  the  behavior  of  casein  in  milk.  The  addition  of  weak 
acids  to  these  salt  solutions  forms  precipitates  of  the  free  nucleo- 
albumin  acids.  From  very  weak  solutions  the  precipitate  may  not 
separate  until  after  heating.  A  large  number  of  bodies  have  been  de- 
scribed as  nucleo-albumins,  but  only  those  will  be  mentioned  here 
which  are  well  known.  In  the  newer  classification  referred  to  above 
all  these  compounds  are  described  simply  as  phospho-proteins.  No 
assumption  is  made  regarding  the  exact  form  in  which  the  phosphorus 
is  held,  but  the  combination  may  be  in  a  general  way  that  of  an  ester 
of  phosphoric  acid. 

Casein.  Of  all  the  nucleo-albumins  this  is  the  best  known  and 
most  important.  It  occurs  in  milk  as  a  neutral  calcium  salt,  and  in 
the  case  of  cow's  milk  makes  up  nearly  4  per  cent  by  weight.  It  may 
be  readily  separated  from  milk  by  the  addition  of  a  little  acetic  acid. 
In  precipitating,  the  fat  is  usually  carried  down  too,  but  may  be  re- 
moved after  drying  by  treatment  with  ether  or  petroleum  spirit. 
Rennin,  a  peculiar  enzyme  of  the  stomach,  to  be  described  later,  causes 
a  kind  of  coagulation  in  casein  solutions;  if  lime  salts  are  present, 
which  is  practically  the  case  in  milk,  the  coagulation  extends  to  the 
formation  of  a  curd  or  cheesy  mass  which  is  very  characteristic.  The 
first  product  formed  by  the  rennin  is  known  as  paracasein  and  the 
curd,  or  cheese,  is  the  calcium  combination  of  this. 

Casein  was  formerly  considered  as  an  alkali  albuminate  because  of 
its  behavior  with  acids  and  alkali  solutions.  Many  of  its  alkali  com- 
binations are  now  produced  in  a  technical  way  as  by-products  in  the 
butter  and  cream  industries.  Plasmon  and  nutrose  are  apparently 
sodium-casein  compounds.  These  are  used  as  foods,  but  some  of  the 
others  find  application  in  other  directions.  Casein  forms  two  series  of 
salts  with  calcium  hydroxide  and  other  bases  and  the  amount  of  metal 
in  several  of  these  has  been  found  with  considerable  accuracy.  Most 
of  these  salts  form  opalescent  rather  than  perfectly  clear  solutions. 
The  addition  of  sodium  chloride  or  magnesium  sulphate  to  these  solu- 
tions in  sufficient  amount  completely  precipitates  the  casein.  Like  the 
other  nucleo-albumins,  casein  leaves  a  pseudo-nuclein  residue  on  di- 
gestion with  pepsin  and  hydrochloric  acid. 

In  combining  casein  with  alkali  1  gram  of  the  former  may  be  dis- 
solved in  4.5  cc.  of  N/10  sodium  hydroxide  or  equivalent  solution. 
But  this  is  still  acid  toward  phenol-phthalein.  To  obtain  a  solution 
neutral  with  phenol-phthalein  just  twice  as  much  alkali  must  be  used. 
The  second  reaction  corresponds  to  an  equivalent  weight  of  nil  for 
the  casein.     Casein  shows  also  a  basic  behavior  and  unites  readily  with 


THE    PROTEIN    SUBSTANCES.  73 

many  acids,  i  gram  combines  with  7  cc.  almost  exactly,  of  N/10 
hydrochloric  or  equivalent  strong  acid,  the  reactions  being  completed 
without  the  aid  of  heat.  These  reactions  illustrate  very  beautifully 
the  chemical  behavior  of  complex  groups  of  amino  acids.  Something 
will  be  said  later  about  the  method  of  preparing  pure  casein  used  in 
such  tests. 

Vitellin.  While  white  of  egg  contains  essentially  albumin  proper 
and  globulin,  the  yellow  part  is  extremely  complex,  containing  many 
substances.  At  least  two  of  these  compounds  hold  phosphorus  in  com- 
bination; one  of  these  is  lecithin,  referred  to  earlier,  and  the  other  is 
the  nucleo-albumin  called  vitellin.  The  separation  of  these  substances 
from  each  other  is  extremely  difficult.  Vitellin  is  not  soluble  in  water, 
but  dissolves  in  weak  alkali  solutions;  on  digestion  with  pepsin  and 
hydrochloric  acid  it  yields  a  pseudo-nuclein  residue  which  contains 
iron  as  well  as  phosphorus.  The  name  hematogen  has  been  given  to 
this,  and  it  is  considered  as  of  great  physiological  importance  because 
of  its  iron  content.  It  is  possibly  one  of  the  parent  substances  of 
hemoglobin. 

Other  Nucleo-albumins.  In  the  eggs  of  fishes  there  is  found  a 
peculiar  vitellin  called  ichthulin,  which  has  been  obtained  in  crystalline 
form.  It  is  not  soluble  in  water,  but  yields  a  clear  solution  with  weak 
alkalies. 

In  cell  protoplasm  several  different  nucleo-albumins  are  found. 
These  bodies  contain  iron,  are  insoluble  in  water  in  pure  condition,  but 
with  alkalies  form  salts  which  are  readily  soluble. 

Vegetable  Proteins.  Most  of  the  protein  bodies  thus  far  referred 
to  have  belonged  to  the  animal  kingdom,  but  as  plant  constituents 
fully  as  great  a  number  occur.  The  exact  nature  of  some  of  these  is 
obscure,  but  many  valuable  observations  have  been  made  by  Osborne 
and  other  chemists  in  the  last  few  years  which  have  cleared  up  some 
of  the  points  in  dispute.    Only  brief  mention  can  be  made  here. 

In  wheat  flour,  for  example,  four  or  five  protein  bodies  appear  to 
be  present.  The  most  abundant  of  these  is  called  by  Osborne  glutenin 
and  makes  up  over  4  per  cent  of  the  weight  of  the  grain.  Next  in 
abundance  is  another  important  compound  known  as  gliadin,  amount- 
ing to  about  4  per  cent  of  the  grain  weight.  These  two  proteins  unite 
in  the  formation  of  gluten  which  is  essential  in  the  production  of  an 
elastic  dough,  which  on  leavening  yields  a  porous  and  light  bread. 
Gliadin  is  soluble  in  dilute  alcohol  and  forms  an  opalescent  solution 
with  water.  In  some  respects  it  resembles  a  globulin.  In  its  behavior 
with  weak  alkalies  glutenin  bears  some  resemblance  to  casein.  Wheat 
flour  contains  also  a  true  globulin  in  small  amount. 


74  PHYSIOLOGICAL    CHEMISTRY. 

A  peculiar  protein  body  known  as  zein,  or  maize  fibrin,  is  found 
in  corn  meal.  It  is  soluble  in  alcohol  but  not  in  water,  and  is  not 
soluble  in  dilute  alkali  solutions.  Corn  contains  also  three  globulin- 
like bodies  and  one  or  more  substances  to  be  classed  with  the  albumins 
proper. 

Legumin  is  found  in  peas,  beans  and  related  seeds ;  it  was  formerly- 
placed  in  the  group  of  nucleo-albumins,  but  in  its  solubility  conditions 
resembles  the  typical  globulins  and  is  now  so  included.  The  legumin 
obtained  from  vetches  does  not  coagulate  on  boiling.  On  boiling  a 
solution  of  pea  legumin  a  jelly-like  substance  is  formed. 

Recently  Osborne  has  proposed  the  nzmtprolamins  for  the  seed  proteins  soluble 
in  alcohol.  As  the  best  representatives  of  this  class  we  have  the  gliadin  of  wheat 
and  the  zein  of  corn,  just  mentioned,  and  the  hordein  from  barley.  These  proteins, 
which  are  soluble  in  all  proportions  in  alcohol  of  70  to  80  per  cent,  are  found  in  the 
seeds  of  all  cereals,  apparently,  and  constitute  a  relatively  large  proportion  of  their 
reserve  material.  They  do  not  appear  to  occur  in  other  parts  of  the  plant.  On 
decomposition  these  prolamins  yield  relatively  large  amounts  of  glutaminic  acid. 

The  glutelins,  according  to  the  same  author,  make  up  a  large  part  of  the  protein 
matter  of  cereals.  They  are  said  to  be  insoluble  in  all  neutral  solvents,  but  dis- 
solve in  weak  acid  or  alkalies.  The  glutenin,  mentioned  above  from  wheat  flour, 
is  the  best  known  member  of  the  group,  because  of  its  ready  accessibility  and  ease 
of  preparation.  It  is  difficult  to  separate  the  glutelins  from  other  seeds  in  a  form 
pure  enough  for  study,  because  they  yield  no  coherent  gluten,  to  begin  with. 

Seeds  contain,  also,  compounds  which  appear  to  be  true  nucleo-proteins,  that  is 
combinations  of  nucleic  acid  with  a  protein  group.  But  the  separation  and  identi- 
fication of  these  bodies  has  not  been,  thus  far,  satisfactorily  carried  out. 

THE   HISTONES. 

These  are  relatively  simple  proteins  which,  apparently,  always  occur 
in  combination  with  certain  groups  to  form  the  nucleo  proteids,  or 
conjugated  proteins.  They  behave  as  rather  strong  bases  and  yield 
basic  groups  on  cleavage.  In  consequence  of  their  basic  character  they 
are  precipitated  from  solution  by  addition  of  alkalies,  especially  by 
ammonia.  In  presence  of  salts  they  are  coagulated  by  boiling,  and  are 
also  precipitated  in  cold  solution  by  nitric  acid;  this  precipitate  disap- 
pears on  warming,  to  return  on  cooling.  They  yield  precipitates  with 
the  alkaloid  reagents  in  neutral  as  well  as  in  acid  solutions.  The 
nitrogen  content  of  the  histones  is  relatively  high  and  the  sulphur 
content  low.  They  contain  no  phosphorus.  Histones  are  obtained 
from  several  sources,  and  the  best  known  are  the  following : 

Globin.  This  makes  up  about  96  per  cent  of  the  hemoglobin  of  the 
red  blood  corpuscle,  existing  in  combination  with  the  iron-containing 
constituent,  hematin.  It  is  precipitated  by  a  relatively  small  amount 
of  ammonia,  and  redissolved  by  a  slight  excess.    On  cleavage  it  yields 


THE    PROTEIN    SUBSTANCES.  75 

much  histidine  and  leucine.  Of  all  the  histones  this  is  the  one  most 
readily  obtained  for  experiment. 

Salmo-histone,  Scomber-histone,  and  Gadus-histone.  These 
bodies  are  obtained  from  the  immature  testicles  of  the  salmon,  the 
mackerel  and  the  codfish,  and  were  first  classed  as  albumoses.  But 
their  precipitation  reactions  throw  them  into  the  group  of  histones. 
Similar  products  have  been  obtained  from  the  testicles  of  other 
animals. 

Nucleo-histone.  This  name  was  given  to  a  product  separated 
from  the  thymus  glands  of  the  calf  and  was  one  of  the  first  studied. 
On  cleavage  it  yields  much  arginine  and  tyrosine,  and  is  characterized 
by  easy  digestibility. 

As  strongly  basic  bodies  the  histones  show  the  interesting  property 
of  forming  precipitates  with  many  of  the  other  simple  albumins, 
especially  with  casein,  egg  albumin  and  serum  albumin.  Their  pre- 
cipitates contain  the  component  proteins  in  definite  proportions. 

PROTAMINES. 

We  come  here  to  the  simplest  of  all  the  naturally  occurring  proteins. 
They  do  not  exist  free  in  nature  but,  like  the  histones,  in  combination 
with  nucleic  acids,  hematin  or  other  simple  "prosthetic  group."  The 
protamines  contain  no  sulphur  but  are  very  rich  in  nitrogen  and  low 
in  carbon  as  compared  with  the  ordinary  proteins.  They  are  not 
coagulated  by  heat  and  do  not  give  the  Millon's  reagent  reaction  or 
that  of  Adamkiewicz.  The  biuret  reaction  is  marked  and  the  alkaloid 
reagents  produce  precipitates.  Some  of  the  groups  in  the  common 
proteins  are  therefore  wanting  in  the  protamines.  Several  of  these 
bodies  have  been  isolated,  particularly  from  the  nucleo-proteids  of 
fish  spermatozoa  and  the  names  given  to  them  suggest  their  origin. 
Thus,  we  have  salmin,  sturin,  scombrin  and  clupein.  In  recent 
analyses  the  following  formulas  have  been  found  for  the  more  im- 
portant protamines : 

Salmin    C80H67N„O, 

Clupein   Ca„H02H14O9 

Scombrin     C32H7:N10O8 

Sturin   C34H7,N17O0 

When  warmed  with  weak  acid,  or  when  subjected  to  pancreatic 

digestion,  they  yield  at  first  proiones,  corresponding  to  the  peptones 

of  ordinary  digestion  and  finally  simpler  products,  among  which  the 

■ne    bases,    arginine,    lysine    and    histidine    predominate.      From 

salmin,  for  example,  over  80  per  cent  of  arginine  has  been  obtained. 


76  PHYSIOLOGICAL    CHEMISTRY. 

In  some  cases  of  decomposition  the  cleavage  into  the  hexone  bases 
has  been  nearly  quantitative,  which  is  an  important  step  toward  estab- 
lishing the  empirical  formula  of  the  parent  protamine.  The  prota- 
mines appear  to  have  rather  marked  toxic  properties. 

The  histones  are  more  complex  bodies  than  the  protamines,  and 
possibly  contain  the  latter  as  a  component  part;  It  is  also  possible  that 
the  histones  represent  a  stage  in  the  development  of  the  protamines, 
since  while  the  former  are  found  in  immature  spermatozoa,  the  latter 
are  commonly  obtained  from  the  mature  organisms.  In  basic  prop- 
erties the  protamines  are  more  marked  than  are  the  histones,  and  are 
precipitated  easily  by  alkalies.  They  do  not  seem  to  be  altered  by 
peptic  digestion,  but  by  trypsin  and  erepsin  they  may  be  reduced  to 
crystalline  products.  The  protones,  referred  to  above,  are  stages  in 
this  cleavage. 

From  a  purely  scientific  standpoint  these  bodies  possess  great  in- 
terest and  importance,  since  they  represent,  apparently,  the  beginnings 
in  the  formation  of  protein  molecules.  On  cleavage  they  yield  groups 
of  amino  acids  which  are  quantitatively  more  readily  measured  than 
are  the  products  from  the  more  complex  proteins. 

TRANSFORMATION    PRODUCTS. 

The  protein  bodies  which  have  been  described  in  the  foregoing 
pages  are  natural  unmodified  substances  or  primary  products.  We  have 
now  to  consider  briefly  a  class  of  important  protein  compounds  which 
includes  secondary  or  modified  substances  which  in  the  main  are  de- 
rived from  the  native  albumins  just  discussed.  These  modified  forms 
may  be  obtained  in  various  ways,  but  for  convenience  three  groups  of 
transformation  products  may  be  made,  as  shown  below. 

COAGULATED   OR   MODIFIED   ALBUMINS. 

It  has  been  shown  already  that  white  of  egg  dissolves  easily  in 
water.  The  solution  so  made  undergoes  a  change  when  heated  or 
when  treated  with  certain  reagents.  This  change  is  called  coagulation 
and  the  resultant  product  is  so  essentially  altered  that  it  may  no  longer 
be  brought  into  the  original  form,  or  a  similar  form,  by  any  known 
means.  Some  of  the  conditions  of  coagulation  have  been  explained 
above  and  illustrated  by  experiments.  While  the  simple  or  native 
egg  albumin  is  soluble  in  water  the  modified  product  is  insoluble.  It 
is,  however,  soluble  in  weak  acids  or  alkalies,  but  is  insoluble  in  solu- 
tions of  neutral  salts.  It  follows,  therefore,  that  while  coagulation  or 
modification  of  a  native  albumin  always  follows  on  heating,  pre  dpi- 


THE    PROTEIN    SUBSTANCES.  77 

tat  ion  may  not  result.  This  depends  on  the  reaction  of  the  mixture. 
Coagulation  or  modification  on  the  one  hand  and  precipitation  on  the 
other  are  perfectly  distinct  phenomena.  In  the  case  of  egg  albumin 
in  solution,  for  example,  a  precipitate  forms  on  heating  as  long  as 
the  solution  is  nearly  neutral.  In  presence  of  salts  the  precipitation 
is  more  complete.  But  if  the  original  solution  is  alkaline  modification 
of  the  albumin  takes  place  but  without  precipitation,  as  soluble  alkali 
albuminate  is  now  formed.  In  presence  of  acid  in  proper  amount 
soluble  acid  albumin  is  formed.  Although  often  used  synonymously 
the  terms  coagulation  and  precipitation  have  here  distinct  meanings. 

The  exact  nature  of  the  change  which  takes  place  when  native  albu- 
mins are  heated  is  not  known.  Hence  the  terms  used  in  describing 
the  phenomenon  are  somewhat  indefinite.  They  are  "  modified,"  or,  to 
freely  render  a  German  expression,  "  denatured."  To  bring  them 
again  into  the  original  condition  is  not  possible.  White  of  egg  may 
sometimes  be  modified  or  altered  without  becoming  opaque,  and  the 
same  is  true  of  clear  blood  serum.  In  both  cases  we  have  coagulation 
without  precipitation. 

Some  of  these  changes  in  condition  of  the  protein  are  termed  re- 
versible, and  others  irreversible.  Many  of  the  precipitation  reactions 
are  reversible;  that  is,  the  protein  may  afterwards  be  returned  to  its 
former  condition.  But  the  change  produced  in  a  protein  by  coagula- 
tion, for  example,  is  irreversible. 

ACID    AND    ALKALI    ALBUMINS. 

These  products  represent  the  most  important  forms  of  the  coagu- 
lated modified  albumin,  and  may  be  looked  upon  as  forming  salts  of 
the  albumin  nucleus  acting  as  an  acid  or  basic  ion.  They  are  most 
readily  secured  by  the  action  of  acid  or  alkali  in  excess  on  some  native 
albumin,  usually  white  of  egg.  These  actions  of  alkali  or  acid  are 
but  the  beginning  of  the  profound  changes  in  which  the  protein  mole- 
cule finally  breaks  down  into  small  groups.  They  may  not  be  looked 
upon,  therefore,  as  absolutely  sharp  and  definitely  limited  conditions, 
which  may  always  be  exactly  duplicated. 

Alkali  Albuminates.  Strong  alkali  solutions  act  very  energetically 
on  white  of  egg  and  the  reaction  is  always  accompanied  by  some  de- 
composition of  the  latter.  There  is  a  loss  of  nitrogen  in  the  form 
of  ammonia,  and  of  sulphur  as  hydrogen  sulphide.  The  reaction  with 
lead  solution,  production  of  lead  sulphide,  disappears  after  the  alkali 
treatment.  The  most  characteristic  product  of  alkali  action  on  native 
albumin  is  a  thick  jelly-like  mass  and  is  known  as  "  Lieberkuehn's 
jelly."    It  may  be  obtained  as  follows : 


78  PHYSIOLOGICAL    CHEMISTRY. 

Experiment.  Add  strong  sodium  hydroxide  solution  to  white  of  egg,  with  con- 
stant stirring,  until  a  thick  jelly  is  formed.  Too  much  alkali  must  not  be  added 
here,  but  just  enough  to  make  the  maximum  of  jelly.  This  is  next  cut  into  small 
pieces  and  washed  in  distilled  water  several  times  until  the  lumps  are  white  through- 
out. They  are  then  heated  with  fresh  pure  water,  but  very  gently,  until  they  go 
into  solution.  This  is  then  filtered  and  the  nitrate  precipitated  by  acetic  acid,  avoid- 
ing any  excess.  The  precipitate  is  washed  with  pure  water,  and  used  for  experi- 
ments below. 

This  precipitate  is  the  modified  alkali  albumin  or  alkali  protein 
proper.  It  is  likewise  insoluble  in  salt  solutions.  In  the  treatment 
with  the  alkali  a  salt  of  the  modified  protein  is  formed,  and  this  is 
called  an  alkali  albuminate.  The  salt  is  readily  soluble,  while  the 
alkali-protein  itself  is  not. 

Experiment.  Use  some  of  the  alkali  albumin  of  the  last  experiment  to  test  other 
properties.  Dissolve  a  portion  in  weak  hydrochloric  or  sulphuric  acid  and  observe 
that  the  solution  does  not  coagulate  on  boiling.  An  acid  solution  is  precipitated 
by  addition  of  sodium  chloride  to  saturation,  and  it  is  also  precipitated  by  adding 
weak  alkali  to  the  point  of  neutrality.  When  this  neutral  point  is  reached  more 
alkali  brings  about  solution  again. 

The  formation  of  Lieberkuehn's  jelly  illustrates  the  production  of 
the  alkali  albumin  at  once  in  the  cold.  A  similar  result  is  obtained  by 
heating  some  white  of  egg  solution  for  a  time  with  very  weak  alkali. 
A  clear  solution  is  finally  obtained. 

Experiment.  Dilute  white  of  egg  with  water  and  add  a  small  amount  of  N/io 
alkali  solution.  A  few  cubic  centimeters  will  suffice.  Keep  the  mixture  at  a  tem- 
perature of  about  400  to  45°  on  the  water-bath  through  an  hour,  and  then  test  some 
of  it  by  boiling  in  a  test-tube.  It  should  not  coagulate.  To  a  portion  of  the 
clear  solution  add  a  few  drops  of  phenol-phthalein  indicator  and  then  run  in  dilute 
sulphuric  acid  to  neutralization.      A  precipitate  forms  as  shown  above. 

Acid  Albumin.  According  to  the  view  held  at  one  time  the  solu- 
tion of  the  alkali  albuminate  in  water  yields  an  acid  albumin  on  acid 
treatment.  But  the  weight  of  evidence  now  indicates  that  the  group 
in  the  albuminate  having  an  acid  function  is  different  from  the  group 
in  the  so-called  acid  albumin  which  certainly  plays  the  part  of  a  basic 
radical.  Although  the  albuminate  and  the  acid  albumin  have  certain 
points  in  common,  as  will  be  shown,  they  are  not  identical.  It  appears, 
however,  that  while  the  albuminate  may  not  be  converted  into  acid 
albumin  by  action  of  weak  acid,  the  opposite  conversion  is  possible; 
that  is,  weak  alkali  will  change  acid  albumin  into  albuminate.  Some 
simple  experiments  may  be  made  here : 

Experiment.  Dilute  white  of  egg  with  four  volumes  of  water,  take  25  cc.  of 
the  mixture,  add  5  cc.  of  0.2  per  cent  hydrochloric  acid  and  warm  it  on  the  water- 
bath  for  about  two  hours  to  a  temperature  of  450  C.  Then  carefully  neutralize 
the   solution   with    dilute   sodium   hydroxide,    using   phenol-phthalein    as    indicator. 


THE    PROTEIN    SUBSTANCES.  79 

This  precipitates  insoluble  acid  albumin,  which  can  be  washed  with  water  by  decan- 
tation.  It  is  essential  that  just  the  right  amount  of  alkali  be  added  here;  an 
excess  would  redissolve  the  precipitated  acid  albumin  with  formation  of  alkali 
albuminate.     The  washed  acid  albumin  can  be  used  for  a  number  of  tests. 

Experiment.  Dissolve  a  little  of  the  washed  acid  albumin  in  water  by  the  aid 
of  weak  hydrochloric  acid,  and  note  that  the  solution  does  not  coagulate  on  boil- 
ing. Observe,  however,  that  the  addition  of  common  salt  to  the  acid  solution 
brings  about  precipitation.  The  same  thing  was  found  to  be  true  with  the  solution 
of  alkali  albuminate  in  acid. 

In  forming  acid  albumin  from  a  native  albumin  the  action  of  the 
weak  hydrochloric  acid  employed  is  much  less  destructive  than  is  the 
action  of  the  alkali  in  producing  albuminate.  The  actual  modification 
of  the  protein  molecule  is  much  less  profound.  Nothing  is  split  off  as 
is  the  ammonia  or  hydrogen  sulphide  in  the  other  case,  and  this  may 
account  for  the  observed  fact  that  the  acid  albumin  may  be  changed 
into  albuminate  by  use  of  weak  alkali.  It  must  of  course  be  remem- 
bered that  a  stronger  acid  may  not  be  used  in  making  the  acid  albumin, 
since  here  too  the  reaction  may  become  destructive. 

Syntonin.  This  appears  to  be  an  acid  albumin,  resulting  from  the 
action  of  dilute  acids  on  muscle,  and  is  very  readily  formed  in  pres- 
ence of  the  ferment  pepsin.  The  name  is  often  applied  to  all  acid 
albumins,  but  it  is  perhaps  preferable  to  restrict  its  use  to  describe  the 
product  from  muscle. 

Experiment.  Free  the  muscle  part  of  meat  from  fat  as  for  as  possible  and  run 
it  through  a  sausage  mill  several  times  to  bring  it  to  a  fine  state  of  subdivision. 
Wash  this  chopped  mass  with  distilled  water  until  the  washings  remain  clear.  Now, 
to  about  s  gm.  of  the  moist  residue  in  a  small  flask,  add  50  cc.  of  dilute  hydro- 
chloric acid,  containing  0.1  per  cent  of  the  true  acid.  Warm  the  mixture  slightly 
(to  350  or  40°  C),  and  keep  at  this  temperature  about  three  hours.  Then  filter 
and  test  the  filtrate.  It  contains  the  soluble  syntonin,  held  by  the  excess  of  weak 
acid  used. 

Experiment.  To  a  small  portion  of  the  filtrate  add  weak  caustic  soda,  which 
produces  a  precipitate  soluble  in  excess  of  the  alkali.  This  latter  solution  contains 
albuminate. 

Boil  another  portion  of  the  filtrate.  It  does  not  coagulate  directly,  but  after 
the  addition  of  common  salt  precipitation   follows. 

It  must  be  remembered  that  the  action  of  both  acids  and  alkalies  on 
the  native  albumins  may  easily  extend  beyond  the  formation  of  the 
simple  products  here  mentioned.  These  are  merely  limiting  cases.  It 
has  been  already  shown  that  by  more  prolonged  action  various  prod- 
ucts of  disintegration  are  obtained  and  the  substances  just  described 
represent  the  first  stages.  With  slightly  stronger  acids  or  alkalies  or 
by  elevation  of  temperature  the  more  easily  separated  of  the  amino 
complexes  begin  to  split  off.  The  condition  of  stability  is  only  relative. 
With  molecules  as  large  as  these  it  may  even  be  possible  to  separate 


80  PHYSIOLOGICAL    CHEMISTRY. 

some  of  the  outlying  groups  without  greatly  impairing  the  integrity 
of  the  whole. 

It  will  be  recalled  that  in  the  second  classification  of  the  protein 
substances,  given  at  the  outset,  a  group  of  so-called  metaproteins  was 
mentioned.  This  group  includes  the  alkali  albumins  and  the  acid 
albumins,  but  not  the  salts.  That  is,  it  does  not  include  the  so-called 
albuminates  or  the  opposite  class  of  bodies,  which  consists  of  combina- 
tions of  acid  proteins  with  acids.  This  distinction  should  be  kept  in 
mind.  It  will  be  recalled,  further,  that  the  less  highly  modified  pro- 
tein, formed  by  the  action  of  water  alone,  is  called  in  this  classification 
a  protean.  The  proteans  are,  like  the  acid  and  alkali  albumins,  in- 
soluble in  water. 

ALBUMOSES   AND   PEPTONES. 

By  the  simple  treatment  with  weak  acids  or  alkalies  alone,  the 
changes  in  the  native  protein  bodies  are  of  the  character  described  in 
the  last  paragraph.  But  in  presence  of  certain  enzymes  further  modi- 
fications are  reached  and  these  have  received  the  names  of  albumoses 
and  peptones  when  they  are  produced  by  the  ferments  of  the  digestive 
tract.  It  is  indeed  true  that  these  substances  may  be  produced  in 
fairly  large  amount  by  the  simple  chemical  treatment  or  by  heating 
the  protein  substances  with  water  under  pressure.  But  the  names,  in 
practice,  are  usually  restricted  to  the  products  of  enzymic  formation. 

Of  the  exact  nature  of  the  reactions  by  which  these  substances  are 
reached  little  is  known.  They  represent  the  very  last  stages  in  the 
process  of  breaking  down  complex  native  protein  bodies  which  still 
give  the  characteristic  protein  tests.  Further  disintegration  leads  to 
bodies  which  are  no  longer  proteins,  but  which,  as  amino  acids,  are 
simply  constituent  groups  of  the  complex  protein  molecule.  The 
peptone  substances  represent  a  more  advanced  stage  of  modification 
than  do  the  albumoses.  In  both  groups  of  bodies  we  find  the  reactions 
with  the  alkaloid  reagents  and  with  the  precipitating  metallic  solutions 
in  most  cases  still  marked;  the  biuret  reaction  is  also  still  present. 
But  for  the  peptones  we  find  lacking  the  property  on  which  the  salting 
out  processes  depend.  By  adding  plenty  of  ammonium  sulphate  or 
zinc  sulphate  it  is  possible  to  throw  the  albumoses  out  of  solution; 
the  peptones  do  not  respond  to  this  treatment  and  in  other  points  also 
they  are  further  removed  from  the  original  proteins  than  are  the 
albumoses. 

But  it  must  not  be  understood  that  the  distinction  between  the  two 
groups  is  perfectly  simple  and  clear.    Unfortunately  much  confusion 


THE    PROTEIN    SUBSTANCES.  ol 

still  prevails  in  the  literature  of  the  subject  and  an  elementary  pre- 
sentation which  is  satisfactory  and  consistent  is  not  yet  possible.  In 
this  chapter  only  a  brief  outline  of  the  relations  now  generally  accepted 
among  chemists  and  physiologists  will  be  attempted,  while  in  a  follow- 
ing chapter  on  digestion  some  of  the  more  practical  details  will  receive 
consideration. 

Basis  of  Classification.  The  general  classification  of  these  sub- 
stances commonly  recognized  is  that  of  Kiihne,  which  was  elaborated 
mainly  in  conjunction  with  Chittenden.  The  scheme  has  been  en- 
larged and  modified  somewhat  by  other  workers  but  in  its  important 
features  the  ideas  of  Kiihne  still  hold  the  first  place.  In  the  weak  acid 
as  well  as  in  the  enzymic  treatment  it  is  easily  seen  that  the  common 
proteins  are  not  homogeneous  or  symmetrical  bodies.  On  the  con- 
trary they  seem  to  contain  two  great  groups  which  respond  very  differ- 
ently to  the  action  of  the  digestive  agent,  whether  acid  or  ferment. 
A  part  of  the  original  complex  appears  to  break  down  rather  quickly 
and  go  into  a  soluble  form ;  while  a  second  portion  resists  this  breaking 
down  process  pretty  effectually  as  far  as  weak  acid  and  pepsin  fer- 
mentation is  concerned,  at  any  rate,  and  in  subsequent  treatment  with 
the  more  active  pancreatic  ferment  it  yields  products  different  from 
those  derived  from  the  first  group.  To  the  first  or  less  resistant  frac- 
tion, Kiihne  gave  the  name  hemi  group,  and  to  the  second  or  more 
resistant  portion,  the  name  anti  group.  It  was  later  noticed  that  most 
protein  bodies  seem  to  contain  a  third  group  which  in  the  subsequent 
breaking  down  yields  a  sugar  of  some  kind.  Hence  a  further  or 
carbohydrate  group  may  be  assumed  to  exist  in  the  native  protein 
molecules,  or  in  most  of  them,  at  least.  But  the  latest  researches  seem 
to  show  that  the  amount  of  this  complex  present  is,  in  most  cases, 
not  large. 

Albumoses.  In  the  first  stage  of  the  action  of  the  acid  and  fer- 
ment on  the  protein  body  a  kind  of  acid  albumin  appears  which  passes 
by  continued  digestion  into  the  next  or  albumose  stage.  Different 
albumoses  seem  to  be  derived  from  the  several  native  proteins,  and 
these  may  be  called,  in  general,  proteoses.  Names  have  also  been 
given  to  them  corresponding  to  their  origin.  We  have,  accordingly, 
fibrinoses,  caseoses,  myosinoses,  globulinoses,  and  so  on.  Several  de- 
grees of  albumose  digestion  are  recognized;  that  is,  bodies  are  pro- 
duced which  behave  differently  on  treatment  of  the  digesting  mixture 
with  precipitating  reagents,  and  we  have,  therefore,  primary  and 
secondary  albumoses.  The  secondary  albumose  stage  represents  a 
more  advanced  condition  of  change  on  digestion  than  does  the  primary 
7 


82  PHYSIOLOGICAL    CHEMISTRY. 

albumose.  Finally,  the  secondary  albumose,  by  prolonged  contact 
with  the  digestive  agents,  passes  into  the  peptone  stage.  Some  idea 
of  the  existence  of  these  three  stages  of  change  may  be  obtained  from 
the  following  experiment  in  which  commercial  peptone  is  taken  for 
illustration.  This  is  a  substance  made  by  the  partial  digestion  of 
fibrin,  gelatin,  serum  and  other  bodies  and  is  not  uniform  or  homo- 
geneous in  structure.  It  contains  representatives  of  the  several  classes 
of  derived  digestion  products. 

Experiment.  Dissolve  about  5  gm.  of  commercial  peptone  in  50  c.c.  of  water 
and  use  small  portions  of  the  solution  for  these  tests :  To  one  portion  add  some 
strong  nitric  acid;  this  produces  a  precipitate.  To  a  second  portion  add  a  little 
copper  sulphate  solution,  which  gives  a  light  greenish  precipitate.  To  a  third  por- 
tion add  a  few  drops  of  acetic  acid  and  then  some  potassium  ferrocyanide.  This 
makes  a  turbidity  or  may  even  cause  a  precipitate.  Now  to  the  remaining  and  large 
portion  of  the  original  solution  add  an  equal  volume  of  a  saturated  solution  of  am- 
monium sulphate.  A  marked  precipitate  of  primary  albumose  separates  and  may 
be  filtered  off  after  a  time.  When  the  liquid  has  all  passed  through  the  filter  note 
that  the  precipitate  may  be  easily  dissolved  by  adding  .  fresh  water,  and  further 
that  this  new  solution  is  not  coagulated  by  boiling.  Note  also  that  the  solution  gives 
a  good  biuret  reaction. 

Experiment.  Use  the  filtrate  from  the  primary  albumose  precipitate  for  a  further 
test.  Add  to  it  powdered  ammonium  sulphate  to  complete  saturation,  that  is,  as 
long  as  the  powder  dissolves  on  thorough  shaking.  Then  add  five  to  ten  drops  of 
a  weakly  acid  solution  of  ammonium  sulphate  (which  may  be  obtained  by  adding 
to  10  cc.  of  saturated  ammonium  sulphate  solution  five  drops  of  concentrated  sul- 
phuric acid).  This  last  treatment  with  the  acid  ammonium  sulphate  gives  a  new 
albumose  precipitate  which  after  a  time  may  be  separated  by  filtration.  Save  the 
filtrate  and  test  the  precipitate  as  follows :  Dissolve  it  in  fresh  water  and  test 
portions  with  copper  sulphate,  nitric  acid  and  the  potassium  ferrocyanide.  These 
reagents  gave  precipitates  with  the  original  peptone  solution,  but  yield  nothing  with 
the  solution  of  the  new  albumose,  which  is  called  secondary  albumose. 

Experiment.  The  filtrate  from  the  secondary  albumose  may  finally  be  tested. 
Add  to  it  an  excess  of  concentrated  sodium  hydroxide  solution  and  then  a  drop 
of  dilute  copper  sulphate  solution.  This  gives  a  purple  red  biuret  color,  showing 
the  presence  of  a  soluble  product  not  precipitated  by  ammonium  sulphate  in  excess. 
This  soluble  product  is  the  peptone,  representing  the  last  stage  of  the  true  digestion. 
This  peptone  gives  no  precipitation  reactions  with  the  reagent  used  above. 

The  first  of  these  fractions,  or  the  primary  albumoses,  may  be  con- 
verted by  further  acid  treatment  or  by  digestion  into  secondary  albu- 
moses no  longer  precipitated  by  half  saturated  ammonium  sulphate. 
By  solution  in  water  and  addition  of  alcohol  it  is  possible  to  separate 
this  primary  albumose  into  two  sub- fractions  which  are  pretty  well 
characterized.  The  first  of  these  is  known  as  heteroalbumose  and  is 
insoluble  in  weak  alcohol,  while  the  second,  or  alcohol-soluble  portion, 
is  called  protalbumose.  The  heteroalbumose  belongs  to  the  above  men- 
tioned anti  group  and  is  further  changed  only  with  difficulty.  The 
protalbumose  belongs  to  the  hemi  group.     It  is  quite  soluble  in  water, 


THE    PROTEIN    SUBSTANCES.  83 

and  in  dilute  alcohol  even  more  soluble.  By  prolonged  peptic  diges- 
tion the  protalbumose  passes  into  the  secondary  albumose  known  as 
dcuteralbumose  A,  and  then  into  peptone  B,  so  called. 

An  enormous  amount  of  labor  has  been  devoted  to  the  study  of  the 
various  fractions  obtained  by  digestion  under  different  conditions,  and 
a  complex  nomenclature  describing  the  products  has  grown  up.  But 
the  value  of  much  of  this  is  now  doubted,  as  there  is  no  great  con- 
stancy in  the  results  secured  by  different  observers.  This  much  is  true, 
however,  that  in  the  earliest  stages  of  digestion  certain  amino  com- 
plexes are  very  readily  split  off,  while  others  are  not.  Tyrosine  and 
tryptophane,  for  example,  separate  relatively  quickly,  and  would  be 
considered  as  belonging  to  the  hemi  group.  On  the  other  hand 
glycine,  phenylalanine  and  proline  separate  slowly  and  should 
be  referred  to  the  anti  group.  But  from  the  present  point  of  view 
the  assumption  of  these  two  groups  is  arbitrary  and  without  real  justi- 
fication. The  classification  based  on  it  need  not  be  further  developed 
in  this  book,  which  must  be  kept  within  elementary  bounds. 

Peptones.  The  amount  of  real  peptone  formed  by  the  pepsin  di- 
gestion is  always  small;  the  large  amount  of  peptone  produced  in  the 
body  is  a  consequence  of  the  action  of  the  pancreas  enzyme  known  "as 
trypsin.  The  peptone  of  gastric  digestion  was  assumed  to  be  a  mixture 
of  products  from  the  hemi  and  anti  groups  and  was  called  ampho- 
peptone.  The  term  antipeptone  is  generally  applied  to  the  final  product 
of  the  energetic  pancreatic  digestion.  Amphopeptone  has  been  ob- 
tained as  a  yellow  powder,  very  soluble  in  water  and  very  hygroscopic. 
It  diffuses  pretty  well  through  parchment  and  has  a  sharp  bitter  taste. 
It  is  not  possible  to  salt  out  the  peptone  from  solution,  but  the  alkaloid 
reagents  give  precipitates,  which  are  soluble  in  excess.  Precipitates 
are  formed  by  solutions  of  several  of  the  heavy  metallic  salts  also,  but 
not  by  copper  salts. 

The  two  forms  of  amphopeptone  which  have  been  described  are 
known  as  amphopeptone  A  and  amphopeptone  B.  The  first  is  insol- 
uble in  96  per  cent  alcohol  and  is  further  characterized  by  giving  a 
strong  reaction  with  the  Molisch  reagent  which  relates  it  to  the  carbo- 
hydrate group.  The  second  is  soluble  in  96  per  cent  alcohol  and  does 
not  give  the  Molisch  reaction.  Both  forms  give  a  strong  biuret 
reaction. 

As  to  the  exact  nature  of  the  antipeptone  referred  to  above,  there  is 
still  much  uncertainty.  This  was  assumed  by  Kiihne  to  represent  the 
final  product  of  pancreatic  digestion,  and  it  was  supposed  that  even 
prolonged  digestion  would  not  change  it  further.     It  was  found  later, 


84  PHYSIOLOGICAL    CHEMISTRY. 

however,  that  various  amino  acids  appear  here  in  considerable  quantity, 
and  that  the  digestion  may  be  carried  so  far  as  to  yield  a  product 
which  no  longer  gives  the  characteristic  biuret  reaction;  that  is,  a 
product  from  which  everything  of  a  really  protein  nature  has  disap- 
peared. This  matter  will  be  more  fully  discussed  in  a  following 
chapter.  The  reactions  described  as  characteristic  of  antipeptone  are 
similar  to  those  for  the  amphopeptone  in  the  main.  A  good  biuret 
reaction  is  obtained  if  the  digestion  is  not  too  prolonged,  and  the  alka- 
loid reagents  give  precipitates  which  are  soluble  in  excess.  Some  of 
the  metallic  salts  precipitate,  but  copper  sulphate  not. 

The  name  kyrine  has  been  given  to  certain  kinds  of  peptones  which 
in  a  marked  degree  resist  the  action  of  hydrolysis  through  pepsin  and 
trypsin,  and  which  are  basic  in  character.  In  some  cases  the  compo- 
nent groups  in  these  kyrines  have  been  determined.  For  example,  a 
kyrine  from  casein  is  apparently  made  up  of  i  molecule  of  arginine,  I 
molecule  of  glutaminic  acid  and  2  molecules  of  lysine. 

In  the  formation  of  the  albumoses  and  peptones  from  native  protein 
molecules  a  large  amount  of  water  is  added;  roughly  the  action  may 
be  compared  to  the  hydration  of  starch,  producing  malt  sugar  and 
finally  glucose.  As  the  original  molecule  is  very  large  the  percentage 
amount  of  water  taken  up  in  the  hydration  is  much  less  than  is  the 
case  in  the  carbohydrate  conversion.  It  is  also  very  interesting  to 
note  that  with  the  progress  of  the  hydration  the  amount  of  hydro- 
chloric acid  which  may  be  held  by  the  product  increases;  the  smaller 
molecules  in  the  aggregate  resulting  from  the  hydration  have  a  much 
greater  capacity  for  combining  with  free  hydrochloric  acid  than  the 
parent  substances  have.  This  question  assumes  considerable  practical 
importance  in  connection  with  the  subject  of  gastric  digestion  and 
acidity  of  the  stomach,  as  will  be  shown  later. 

It  must  be  remembered  that  the  various  commercial  products  sold 
as  "  peptones  "  may  contain  many  other  substances,  and  may  be  quite 
unfit  for  use  as  a  food  or  in  medicine.  While  in  some  cases  a  con- 
siderable portion  of  real  peptone  (with  albumose)  is  present,  in  others 
the  main  constituents  are  decomposition  products  formed  by  too  long 
digestion  of  the  meat  or  fibrin  with  the  acid  and  pepsin  mixture. 
Some  of  these  commercial  peptones  appear  to  be  formed  by  digesting 
with  weak  acid  under  pressure,  which  results  in  the  formation  of  bodies 
of  little  nutritive  value;  indeed,  it  is  likely  that  such  products  are  dis- 
tinctly harmful  when  taken  into  the  stomach  of  man. 

It  should  be  mentioned  further  that  many  artificial  products  are 
known  which  are  related  in  properties  to  the  peptones  of  advanced 


THE    PROTEIN    SUBSTANCES.  05 

hydrolysis.     These  may  be  called  peptides,  or  polypeptides.     Some- 
thing will  be  said  about  them  later. 

THE    PROTEIDS. 

The  term  proteid,  as  already  explained,  is  used  to  designate  a  certain 
group  of  protein  compounds.  This  use  is  a  perfectly  arbitrary  one  as 
the  word  was  once  employed  to  describe  all  the  bodies  discussed  in  this 
chapter.  It  would  be  perhaps  well  to  drop  the  term  proteid,  and 
describe  the  bodies  included  under  it  as  conjugated  or  compound  pro- 
teins. According  to  the  generally  accepted  modern  classifications  the 
bodies  now  called  proteids  are  compound  substances  in  which  a  true 
or  native  albumin  is  found  in  combination  with  some  other  group 
which  often  may  be  separated  as  such.  In  the  table  given  earlier  in 
the  chapter  three  such  combinations  are  mentioned  :  the  nucleo-proteids, 
the  hemoglobins  and  the  gluco-proteids.  A  brief  description  of  each 
group  will  here  follow. 

NUCLEO-PROTEIDS. 

These  proteins  are  important  as  making  up  a  large  part  of  the  cell 
nucleus.  In  treating  tissues  rich  in  cells  with  the  pepsin-hydrochloric 
acid  digestive  mixture  it  was  long  ago  recognized  that  a  certain  portion 
went  easily  into  solution,  while  another  portion  was  always  left  undis- 
solved. This  residue  was  called  nuclein  and  was  found  to  contain  all 
the  phosphorus  of  the  original  protein.  If  in  place  of  the  pepsin  mix- 
ture some  other  hydrolyzing  agent  is  used  the  general  result  is  similar ; 
a  separation  into  two  component  parts  takes  place,  and  one  of  these 
parts  is  a  simple  native  protein  substance  and  the  other  the  nuclein  or 
further  and  final  decomposition  product,  nucleic  acid.  The  nucleo- 
proteids  are  therefore  described  as  combinations  of  native  albumins 
with  nucleic  acid. 

In  breaking  down  the  complex  nucleo-proteid  it  appears  that  several 
stages  must  be  distinguished,  the  body  described  as  a  "  nuclein  "  con- 
taining still  some  native  albumin.  Finally,  however,  the  residue  or 
characteristic  part,  the  nucleic  acid,  is  left.  Although  many  investi- 
gations have  been  made  there  is  still  much  uncertainty  about  the  nature 
of  this  acid.  Indeed,  from  different  parent  substances  acids  of  some- 
what different  properties  have  been  obtained,  so  that  it  is  customary  to 
speak  of  the  nucleic  acids.     These  will  be  considered  below. 

Like  the  native  proteins  already  described  the  nucleo-proteids  are 
coagulated  by  heat  and  by  acids.  They  are  soluble  in  water,  salt  solu- 
tions and  also  in  alkali  solutions.     By  means  of  large  excess  of  salt 


86  PHYSIOLOGICAL    CHEMISTRY. 

they  suffer  precipitation.  In  the  last  few  years  nucleo-proteids  from 
different  sources  have  been  studied,  especially  from  yeast  cells,  the 
thyroid  gland,  the  pancreas  and  different  kinds  of  spermatozoa.  The 
sperm  and  spermatozoa  of  sea  urchins  and  fish  have  furnished  a  num- 
ber of  these  substances  because  of  their  relative  richness  in  cell  struc- 
tures. Thus,  characteristic  products  have  been  obtained  from  the 
spermatozoa  of  the  salmon,  the  mackerel,  the  sturgeon  and  so  on. 
These  appear  to  be  distinct  bodies,  but  more  exact  investigations  may 
show  that  the  apparent  differences  depend  on  foreign  proteins  not 
completely  separated  in  their  preparation. 

As  intimated  above,  the  nucleo-proteids  are  found  characteristically 
in  the  organs  rich  in  cells.  The  larger  part  of  the  solid  portion  of 
certain  glands  and  of  the  heads  of  spermatozoa  consist  of  these  conju- 
gated bodies.  The  thymus  of  the  calf  has  been  frequently  used  in  the 
investigations  of  these  bodies,  as  over  75  per  cent  of  the  dried  cells  of 
these  glands  consist  of  a  nucleo-histone.  Fish  spermatozoa  are  easily 
obtainable  from  hatcheries  and  have,  perhaps,  furnished  the  main 
material  for  investigation.  The  dry,  fat-free  portion  of  the  heads, 
which  are  easily  separated,  contains  95  per  cent,  or  more,  of  protamine 
or  histone  combinations  of  nucleic  acids.  The  abundance  of  these 
"  nucleates  "  in  cell  structures,  especially  in  young  cells,  shows  their 
great  physiological  importance.  The  nucleo-proteids  of  various  organs 
have  been  investigated  in  recent  years,  but  the  details  can  not  be 
explained  in  an  elementary  book. 

Iron  seems  to  be  contained  in  the  so-called  "  masked  "  or  non-ionic 
condition  in  the  nucleo-proteids.  It  can  not  be  recognized  by  the  usual 
qualitative  tests,  because  of  its  peculiar  organic  combination.  Iron  in 
this  form  has  long  been  supposed  to  be  important  in  the  formation  of 
red  blood  corpuscles,  which  contain  hematin.  Special  methods  have 
been  devised  for  showing  the  organic  iron. 

The  Nucleic  Acids.  The  occurrence  of  these  important  compounds 
in  combination  with  protamines,  histones  and  other  proteins  has  been 
referred  to  several  times  in  the  last  few  pages.  They  constitute,  in 
fact,  the  important  part  of  the  nucleo-proteids.  By  different  processes 
of  separation  a  number  of  these  acids  have  been  obtained  from  various 
cell  structures,  and  especially  from  yeast  and  fish  sperm  or  sperma- 
tozoa. The  results  of  analyses  lead  to  formulas  of  about  the  follow- 
ing character  in  nearly  all  cases :  C40H52N14O25P4.  These  acids  have 
not  been  obtained  in  crystalline  condition.  They  are  but  slightly  sol- 
uble in  cold  water,  but  soluble  in  weak  alkali  solutions  when  they  form 
salts.     A  number  of  salts  of  the  heavy  metals,  which  are  insoluble  in 


THE    PROTEIN    SUBSTANCES.  87 

water,  have  been  made  and  studied.  When  boiled  in  aqueous  or  acid 
solution  the  nucleic  acids  break  up,  yielding  finally  the  characteristic 
basic  bodies,  long  known  as  the  purine  bases,  the  pyrimidine  bases, 
phosphoric  acid  and  certain  carbohydrates. 

Attempts  have  been  made  to  establish  the  structural  formula  of  some 
of  the  nucleic  acids,  but  without  much  success,  as  they  are  evidently  of 
complex  composition.  Among  the  purines  the  following  have  been 
separated : 

Xanthine  C5H4N402 

Hypoxanthine  C5H4N40 

Adenine  C6H5N5 

Guanine  C5H5N50 

Three  pyrimidine  derivatives  are  known: 

Uracil  C4H4N202 

Cytosine  C4H5N30 

Thymine  C5H6N202 

More  will  be  said  later  about  the  relations  of  these  bodies  to  each 
other  and  to  the  uric  acid  of  the  urine.  The  first  are  important  from 
that  standpoint,  as  in  structure  they  are  closely  related  to  uric  acid,  and 
may  be  forerunners  of  it. 

From  the  amount  of  phosphorus  and  nitrogen  found  by  analysis  of 
the  nucleic  acids  and  the  amount  of  the  bases  secured  on  cleavage,  it 
has  been  suggested  that  they  may  be  complex  esters  of  4  molecules 
of  phosphoric  acid,  in  which  different  bases  may  be  combined.  This 
would  explain  the  existence  of  a  large  number  of  closely  related  acids. 
It  is  not  necessary  to  give  here  the  numerous  empirical  formulas  which 
have  been  suggested  for  the  acids  from  different  sources.  The  typical 
one  given  above  is  sufficient  as  an  illustration  of  the  general  com- 
plexity. Nucleic  acids  from  yeast  and  other  sources  have  found  some 
application  in  medicine.  The  acids  from  other  vegetable  sources  have 
been  studied,  especially  by  Osborne. 

As  acids  these  bodies  have  rather  marked  properties;  they  combine 
not  only  with  inorganic  bases,  but  also  with  simple  proteins  and  many 
toxin  bodies.  The  medicinal  uses  depend  on  these  facts.  The  free 
acids  are  rather  easily  hydrolyzed  by  water  and  mineral  acids,  but  are 
stable  with  alkalies.  The  salts  with  sodium  and  potassium  form  stiff 
jellies  when  dissolved  in  water  by  aid  of  heat,  and  then  cooled  below 
certain  temperatures. 

HEMOGLOBINS. 

The  discussion  of  the  important  subject  of  hemoglobins  may  prop- 
erly be  left  to  be  taken  up  with  the  study  of  the  blood  in  which  ^hey 


88  PHYSIOLOGICAL    CHEMISTRY. 

are  contained.  The  term  is  used  here  in  the  plural  since  from  different 
kinds  of  blood  bodies  of  somewhat  different  properties  have  been 
obtained.  Hemoglobin  in  general  must  be  classed  among  the  com- 
pound bodies  because  it  is  distinctly-  made  up  of  two  characteristic 
parts,  a  histone,  already  referred  to,  and  hematin. 

GLUCO-PROTEIDS. 

We  have  here  a  group  of  bodies  containing  a  number  of  important 
members  about  which  our  knowledge  in  most  cases  is  not  very  ex- 
tended or  exact.  As  the  name  indicates  the  proteins  here  concerned 
contain  a  carbohydrate  constituent  which  may  be  recognized  by  its 
reducing  properties  when  the  substance  in  question  is  warmed  with  a 
weak  acid  and  afterwards  treated  with  Fehling's  solution  in  the  usual 
way.  The  carbohydrate  group  separated  appears  to  be,  in  most  cases 
at  any  rate,  glucose  amine.  Familiar  illustrations  of  these  gluco- 
proteids  are  found  in  the  mucins  and  related  bodies  called  mucoids. 
As  a  class  these  substances  are  characterized  by  relatively  low  nitrogen 
and  high  oxygen  content,  due  to  the  presence  of  the  carbohydrate 
group.  The  amount  of  carbon  present  is  also  lower  than  in  the  com- 
mon proteins.  Of  the  exact  nature  of  the  albumin  combined  with  the 
carbohydrate  little  is  known,  because  in  separating  the  two  groups  by 
acid  or  alkali  treatment  the  protein  constituent  is  so  changed  that  no 
safe  conclusion  can  be  drawn  as  to  its  original  nature. 

The  gluco-proteids  behave  as  acid  bodies.  They  are  not  coagulated 
by  heat  alone,  but  heating  with  acids  or  alkalies  produces  a  complete 
alteration.  With  weak  acetic  acid  a  precipitate  is  in  most  cases  formed 
which  is  not  easily  soluble  in  excess. 

Mucins.  These  bodies  are  found  in  various  secretions,  especially  in 
the  saliva,  bile,  vaginal  fluid,  tears,  nasal  mucus,  etc.  The  amount 
present,  however,  is  always  small  and  the  separation,  in  pure  condition, 
very  difficult.  The  mucin  of  the  submaxillary  gland  is  probably  the 
best  known. 

These  bodies  contain  one  of  the  complex  protein  groups,  since  they 
give  the  reaction  with  Millon's  reagent,  the  xanthoproteic  and  the 
biuret  reactions.  They  are  only  slightly  soluble  in  water  and  in  pres- 
ence of  alkali  produce  a  viscous  stringy  liquid  which  is  extremely 
characteristic,  even  in  great  dilution.  On  warming  with  dilute  alkali 
the  viscous  condition  disappears  through  formation  of  alkali  albumi- 
nate. On  treatment  with  strong  alkali  or  superheated  steam  a  peculiar 
body  is  formed  which,  from  its  discoverer,  is  known  as  Landwehr's 
animal  gum.     This  is  now  known  to  contain  the  protein  and  carbohy- 


THE    PROTEIN    SUBSTANCES.  89 

drate  complexes;  after  diluting  with  weak  acid  and  boiling,  the  sugar 
reaction  may  be  easily  obtained. 

The  mucins  are  much  more  resistant  than  the  nucleo-proteids  against 
the  action  of  reagents  or  ferments,  but  they  undergo  both  peptic  and 
pancreatic  digestion  slowly.  In  urine  the  identification  of  mucin  is 
often  a  matter  of  importance,  as  it  is  frequently  mistaken  for  albumin. 
The  detection  of  mucin  depends  on  the  behavior  with  cold  dilute  acetic 
acid  and  also  on  the  solubility  in  hot  water  after  precipitation  with 
strong  alcohol.     Albumin  is  permanently  coagulated  but  mucin  not. 

Mucoid  Bodies.  These  substances  are  found  in  the  tendons,  carti- 
lage, the  vitreous  body  of  the  eye,  the  cornea  and  elsewhere,  and  are 
closely  related  to  the  mucins.  They  have  the  viscous  properties  of  the 
latter  but  in  general,  in  concentrated  condition,  form  stiffer  jelly-like 
masses.  The  cornea  and  sclerotic  coat  of  the  eye  are  made  up  largely 
of  mucoids  and  collagen  dissolved  in  water. 

The  mucoids  from  tendon  have  been  the  most  thoroughly  studied. 
An  extract  is  made  by  prolonged  treatment  with  weak  lime-water. 
The  solution  is  precipitated  by  acetic  acid  and  the  precipitate  taken  up 
with  ammonia.  These  operations  repeated  several  times  give  a  nearly 
constant  product.  The  analyses  show  48-49  per  cent  of  carbon,  30  of 
oxygen  and  below  12  of  nitrogen.  A  small  amount  of  sulphur  is 
present. 

In  cartilage,  along  with  collagen  and  albuminoid  bodies,  a  very  im- 
portant mucoid  known  as  chondro-mucoid  is  found.  This  has  a  com- 
position not  very  different  from  the  tendon  product  just  given,  but 
contains  over  2  per  cent  of  sulphur,  part  of  which  is  in  peculiar  ethereal 
combination.  This  ethereal  product  is  separated  by  cleavage  with 
dilute  acids  or  alkalies  and  is  known  as  chondroitin  sulphuric  acid,  and, 
according  to  Schmiedeberg,  has  the  composition  Ci8H27N014.S03. 
On  hydrolysis  this  acid  yields  chondroitin,  C18H27N014,  and  sul- 
phuric acid ;  the  chondroitin  furnishes  acetic  acid  and  chondrosin, 
C12H21NOn;  finally  further  hydrolysis  breaks  the  chondrosin  down 
into  glucoseamine  and  glucoronic  acid,  according  to  the  same  author, 
but  later  researches  seem  to  indicate  that  the  cleavage  is  not  as  simple 
as  suggested.  It  has  been  shown  also  that  this  complex  acid  is  not 
peculiar  to  cartilage,  but  is  found  in  many  substances  belonging  to  the 
albuminoid  group  of  proteins  as  well.  Although  widely  distributed 
the  physiological  importance  of  the  body  has  not  yet  been  determined. 

In  addition,  mucoid  substances  have  been  recognized  in  urine,  in 
blood  serum,  in  white  of  egg  and  several  pathological  transudates  in 
small  amount. 


go  PHYSIOLOGICAL    CHEMISTRY. 

ALBUMOIDS  OR  ALBUMINOIDS. 

These  substances  differ  from  the  real  proteins  both  physically  and 
chemically ;  the  physical  differences  are,  however,  the  most  pronounced 
and  characteristic.  The  second  general  classification  of  proteins  places 
these  bodies  in  the  group  of  simple  proteins,  that  is,  they  are  treated 
as  true  proteins.  The  important  bodies  grouped  here  contain  the  dif- 
ferent kinds  of  gelatin  or  glue-forming  compounds,  the  horn  sub- 
stances, the  spongin  of  the  sponge,  elastin  of  the  so-called  elastic 
tissues  of  the  body  and  other  substances  of  less  importance.  They 
are  all  firmer  and  harder  than  the  common  proteins  and  as  a  rule  quite 
insoluble  in  water,  and  in  general  resistant  against  the  action  of  rea- 
gents. While  by  prolonged  treatment  with  superheated  steam  or  acids 
or  alkalies  they  yield  most  of  the  cleavage  products  described  as  char- 
acteristic of  the  albumins,  some  are,  however,  lacking.  The  tyrosine 
group,  for  example,  is  absent  from  gelatin,  or  present  in  minute 
amount  at  most. 

In  food  value  the  albuminoids  are  quite  distinct  from  the  other 
proteins.  Most  of  these  substances  are  so  insoluble  in  the  digestive 
fluids  that  really  no  importance  as  foods  could  be  ascribed  to  them. 
Collagen,  which  yields  gelatin,  has  a  limited  food  value  of  a  peculiar 
kind  which  will  be  referred  to  below.  All  these  substances  serve  as 
supporting,  connecting  or  protective  tissues  in  the  body,  and  they  are 
characterized  necessarily  by  a  kind  of  permanence,  which  depends  on 
insolubility  in  the  first  degree.  With  increasing  age  of  the  body  the 
albuminoid  tissues  become  harder,  firmer  and  less  elastic. 

COLLAGEN. 

The  best  known  of  all  these  albuminoids  is  the  collagen,  or  glue- 
forming  substance,  found  as  ossein  in  bone,  in  cartilage,  in  the  fibrils 
of  connective  tissue,  in  tendons,  in  fish  scales  and  elsewhere.  This 
substance,  wherever  found,  is  insoluble  in  cold  water,  but  by  pro- 
longed heating  with  water  it  passes  into  the  soluble  form  known  as 
gelatin,  glutin  or,  in  impure  condition,  as  glue.  The  change  seems  to 
depend  on  the  taking  up  of  a  molecule,  or  more,  of  water.  At  the 
present  time  it  is  made  in  enormous  quantities  from  slaughter  house 
by-products  and  according  to  its  purity  is  employed  for  different  pur- 
poses. When  made  by  hot  water  extraction  from  clean  bones  or  car- 
tilage it  is  used  as  an  adjunct  to  food  and  also  in  the  preparation  of 
emulsions  for  photographic  plates  or  gelatin  paper.  The  product  from 
common  material  is  used  as  joiner's  glue. 

Gelatin  softens  and  dissolves  in  water  at  a  temperature  above  300. 


THE    PROTEIN    SUBSTANCES.  9 1 

But  this  solution  point  depends  largely  on  the  treatment  to  which  it 
has  been  previously  subjected.  By  long  heating  with  water,  and  espe- 
cially under  the  action  of  superheated  steam  gelatin  gradually  breaks 
down  into  the  usual  cleavage  products  of  the  proteins.  As  this  cleav- 
age progresses  a  point  is  finally  reached  where  the  mixture  no  longer 
solidifies  on  cooling;  a  permanent  liquid  solution  is  obtained.  By 
hydrolysis  with  acids  this  condition  is  much  sooner  reached.  Many 
bacteria  also  have  the  power  of  "  liquefying  "  gelatin,  which  depends 
of  course  on  their  ability  to  decompose  the  complex  into  the  more 
easily  soluble  amino  acids  and  other  compounds. 

Among  the  final  cleavage  products  of  gelatin  easily  recognizable 
glycocoll  and  glutaminic  acid  are  probably  the  most  abundant.  Leu- 
cine, alanine  and  various  other  amino  acids  are  found  in  smaller 
amount.  Like  other  proteins  gelatin  yields  in  peptic  or  tryptic  diges- 
tion bodies  which  have  been  called  gelatoses,  gelatin  peptones  and  so 
on.  These  resemble  but  are  not  identical  with  the  true  peptones,  which 
fact  has  some  bearing  on  the  long-discussed  question  of  the  food  value 
of  gelatin.  Gelatin  is  not  converted  into  true  protein  in  the  animal 
body  and  for  this  reason  cannot  wholly  replace  the  albumins  as  food. 
But  to  some  extent  it  has  the  power  of  protecting  the  so-called  circu- 
lating albumin  from  katabolism,  by  undergoing  destruction  itself. 
This  sparing  or  protecting  power  is  limited,  however,  and  the  gelatin 
substances  can  not  permanently  replace  the  native  proteins  in  this  way. 

Experiments  to  Illustrate  Properties  of  Gelatin.  Dissolve  enough  gelatin 
in  hot  water  to  make  a  solution  of  about  one-half  per  cent  strength.  Use  portions 
of  this  for  tests : 

To  some  of  the  solution  add  a  solution  of  tannic  acid;  this  gives  a  buff  colored 
precipitate.     Gelatin  solution  is,  conversely,  employed  as  a  test  for  tannic  acid. 

To  some  of  the  solution  add  an  excess  of  strong  alcohol;  this  causes  precipitation. 
This  behavior  is  of  importance  in  the  estimation  of  gelatin. 

Use  some  of  the  solution  with  the  test  reagents.  Apply  Millon's  reagent,  the 
biuret  test  and  the  xanthoproteic  test. 

Prepare  a  strong  solution  of  gelatin  in  hot  water.  To  some  of  this  add  solution 
of  potassium  dichromate  and  pour  the  mixture  out  to  cool  in  a  thin  layer  exposed 
to  sunlight.  This  treatment  produces  an  insoluble  mass  which  is  not  attacked  by 
hot  water.    This  property  finds  application  in  photo-engraving  processes. 

To  more  of  the  strong  gelatin  solution  add  a  trace  of  alkali  to  neutralize  any 
acidity  and  then  some  formaldehyde.  On  evaporating  to  dryness  a  hard  mass  is 
obtained  which  is  quite  insoluble  in  water  hot  or  cold  and  which  has  found  many 
applications  in  the  arts. 

Gelatin  to  be  used  in  cooking  should  be  nearly  white  and  should  dis- 
solve in  hot  water  to  form  a  practically  colorless,  odorless  solution. 
Inferior  gelatin  gives  off  a  bad  odor  when  heated  with  water. 

Isinglass  is  a  kind  of  collagen  made  from  the  swimming  bladder  of 


92  PHYSIOLOGICAL    CHEMISTRY. 

certain  large  fishes.  On  heating  with  water  it  yields  a  peculiar  gelatin 
which  dissolves  completely.  Common  isinglass  is  largely  used  in  clari- 
fying beer  and  wine,  while  the  pure  white  varieties  are  employed  in 
thickening  soups  and  jellies. 

KERATIN. 

This  is  the  important  insoluble  substance  in  horn,  the  hoofs  of  cattle, 
finger  nails,  hair  and  feathers.  As  can  be  inferred  from  the  condi- 
tions under  which  it  exists,  it  is  not  easily  attacked  by  water  hot  or 
cold,  by  weak  acids  or  alkalies,  or  by  digestive  fluids.  By  prolonged 
action  of  hot  hydrochloric  acid,  however,  it  undergoes  gradual  hydro- 
lysis and  cleavage  with  formation  of  the  usual  amino  acids  and  other 
products.  Leucine  is  apparently  the  most  abundant  of  these  products, 
as  much  as  18  per  cent  of  the  weight  of  the  horn  shavings  taken  having 
been  obtained  by  certain  investigators.  Other  important  cleavage 
products  found  are  tyrosine,  a-aminoisovaleric  acid,  aspartic  acid,  glu- 
taminic  acid,  phenylalanine,  a-pyrrolidine-carboxylic  acid,  glycocoll,  etc. 

All  keratin  bodies  contain  large  amounts  of  sulphur;  some  of  this 
is  easily  split  off  in  the  form  of  hydrogen  sulphide.  A  large  part 
of  the  sulphur  appears  to  be  present  in  the  complex  body  cystin, 
C6H12N2S204.  The  xanthoproteic  and  Millon's  reagent  reactions  are 
very  characteristic  with  horn  substance.  The  behavior  of  a  drop  of 
nitric  acid  on  the  finger  nail  is  well  known.  The  reactions  with  alka- 
line lead  solutions,  yielding  lead  sulphide,  are  easily  obtained.  The 
use  of  lead  salts  in  hair  dyes  depends  on  this  behavior. 

ELASTIN. 

Elastin  differs  from  keratin  mainly  in  its  higher  content  of  carbon 
and  low  sulphur  content.  In  their  behavior  toward  reagents  they  are 
much  alike.  Like  keratin,  elastin  can  be  dissolved  only  by  change  in 
composition.  Leucine  is  produced  in  large  amount  by  the  hydro- 
chloric acid  cleavage,  and  glycocoll,  tyrosine  and  other  amino  products 
in  smaller  amount.  Subjected  to  peptic  and  pancreatic  digestion 
elastin  is  slowly  dissolved,  yielding  albumins  and  a  kind  of  peptone. 
Most  of  the  protein  reactions  may  be  obtained  from  elastin  after  bring- 
ing it  into  solution  with  alkali. 

AMYLOID    SUBSTANCE. 

This  is  a  body  which  is  found  in  the  so-called  amyloid  degeneration 
of  the  liver  and  kidney.  It  is  particularly  characterized  by  the  reddish 
brown  color  it  assumes  when  heated  with  a  solution  of  iodine  in  potas- 


THE    PROTEIN    SUBSTANCES. 


93 


sium  iodide.  The  analysis  of  amyloid  shows  a  large  amount  of  carbon 
and  some  sulphur.  It  is  insoluble  in  cold  water,  but  partly  soluble  by 
long  heating.  It  gives  the  usual  protein  reactions  when  brought  into 
alkaline  solution,  and  contains  also  a  complex  group  which  yields 
chondroitin  sulphuric  acid. 

FOOD    STUFFS. 

In  the  preceding  pages  the  individual  substances  used  as  foods  or 
occurring  as  essential  principles  of  the  animal  body  have  been  briefly 
discussed.  In  nature  these  compounds  do  not  occur  in  the  pure  free 
condition,  but  are  practically  always  mixed  with  other  compounds. 
Before  passing  to  the  subject  of  digestion  it  will  be  necessary  to  have 
some  idea  of  the  general  composition  of  the  ordinary  foods  as  used 
by  man.  This  information  will  be  presented  in  tabular  form,  the 
figures  being  average  values  from  tables  of  Atwater.  The  fuel 
values  are  given  in  so-called  large  calories. 

ANIMAL    FOODS. 


Loin  of  beef,  edible  portion 

Flank  of  beef,  edible  portion.. 

Ribs  of  beef,  edible  portion 

Round  of  beef,  edible  portion.. 

Canned  corned  beef 

Canned  roast  beef   

Breast  of  veal,  edible  portion.. 
Leg  of  veal,  edible  portion.... 
Leg  of  lamb,  edible  portion.... 
Leg  of  mutton,  edible  portion.. 

Lean  ham,  edible  portion 

Fat  ham,   edible  portion    

Loin   of   pork,   edible   portion.. 

Chicken,  edible  portion 

Turkey,  edible  portion   

P.lack  bass,  edible  portion 

Catfish,  edible  portion    

Salmon,  edible  portion   

Trout,  edible  portion  

Oysters   

Hens'  eggs,  edible  portion  

Butter  

Cheese,  full,  American  

Lard,  unrefined   

Oleomargarin    

Gelatin    


Water 

Protein 

Fat 

Ash 

Per  Cent. 

Per  Cent. 

Per  Cent. 

Per  Cent. 

6l.3 

19.0 

19. 1 

I.O 

59-3 

19.6 

21. 1 

0.9 

57-0 

17.8 

24.6 

0.9 

67.8 

20.9 

10.6 

I.I 

51.8 

26.3 

18.7 

4.0 

58.9 

25-9 

14.8 

1-3 

68.5 

20.4 

10.5 

1.1 

71.7 

20.7 

6.7 

i.l 

58.6 

18.6 

22.6 

1.0 

55-0 

17.3 

27.1 

0.9 

60.0 

25.0 

14.4 

1-3 

38.7 

12.4 

50.0 

0-7 

50.7 

16.4 

32.0 

0.9 

74-8 

21.5 

2.5 

I.l 

55-5 

21. 1 

22.9 

1.0 

76.7 

20.6 

i-7 

1.2 

64.1 

14.4 

20.6 

0.9 

64.6 

22.0 

12.8 

1.4 

77-8 

19.2 

2.1 

1.2 

834 

8.8 

2.4 

1-5 

73-7 

134 

10.5 

1.0 

II.O 

1.0 

85.0 

3-o 

31.6 

28.8 

35-9 

3-4 

4.8 

2.2 

94-0 

0.1 

9-5 

1.2 

83.0 

6.3 

13.6 

91.4 

0.1 

2.1 

Fuel  Value 
in  Calories 
per  Pound. 

1 155 
1255 
1370 

835 
I28o 
1 105 

820 

67O 
1300 
1465 
1075 

2345 
1655 

505 
1360 

455 
1 135 

950 

445 

335 

720 
3605 
2055 
4010 
3525 
1705 


In  the  above  table,  it  will  be  observed,  the  animal  foods  contain  all  a 
large  amount  of  water.  The  solids  consist  essentially  of  proteins  and 
fats.     In  the  vegetable  foods  the  water  is  much  less;  in  most  of  them 


94 


PHYSIOLOGICAL    CHEMISTRY. 


carbohydrates  are  the  characteristic  principles  present.     The  protein 
is  generally  much  lower  than  in  the  animal  foods. 


VEGETABLE    FOODS. 


Corn-,  whole   

Cornmeal    

Popcorn    

Oatmeal   

Rice     

Rye  flour , 

Wheat  flour,   entire 

Wheat  flour,  California  

Wheat  flour,  general  average.. 

White  bread,  wheat 

Whole  wheat  bread  , 

Crackers    

Beans,   dry 

Beans,  dry,  Lima   

Peas,  dry  

Peas,  green,  edible  

Corn,  green,  edible 

Cabbage,  edible  portion   , 

Egg  plant   .  _. 

Potatoes,  edible  portion   

Squash,  edible  portion   

Apples,  edible  portion    , 

Bananas,  edible  portion , 

Chestnuts,  edible  portion  

Hickory  nuts,  edible  portion   . . 
Peanuts,  edible  portion  


15-0 
12.5 

4-3 

7-3 
12.3 
12.9 
11.4 
13.8 
12.0 
35-6 
38.4 

7-1 
12.6 
10.4 

9-5 
74-6 
75-4 
91.5 
92.9 

78.3 

88.3 

84.6 

75-3 

5-9 

3-7 

9.2 


•S  g 


8.2 
9.2 

10.7 

16.1 
8.0 
6.8 

13.8 
7-9 

1 1.4 
9-3 
9-7 

10.2 

22.5 

18. 1 

24.6 

7.0 

3-1 

1.6 

1.2 

2.2 

1.4 

0.4 

1.3 

10.7 

15-4 

25.8 


3-8 

1.9 
5-o 
7.2 

0.3 
0.9 
1.9 
1.4 
1.0 
1.2 
0.9 
8.8 
1.8 
i-5 
1.0 
o.S 
1.1 

0.3 
0.3 
0.1 
0.5 
0.5 
0.6 
7.0 
67.4 
38.6 


-2"    Ph 


68.7 
75-4 
78.7 
67.5 
79.0 
78.7 
71.9 
76.4 
75-i 
52.7 
49-7 
72.4 
59-6 
65-9 
62.0 
16.9 
19.7 
5.6 
5-1 
18.4 
9.0 
14.2 
22.0 
74.2 
1 1.4 
24.4 


fa   u 


1.9 

1.0 

1.4 
0.9 

0.2 

0.4 
O.9 

0-3 
0.5 

1.2 
0.4 

4.4 

4-5 
1-7 
0-5 
I.I 

0.8 
0.4 
0.8 
1.2 
1.0 

2.7 
2.5 


1.4 

1.0 

i-3 
1.9 
0.4 
0.7 
1.0 

0.5 
0.5 

1.2 

i-3 
1.5 
3-5 
4.1 
2.9 
1.0 
0.7 
1.0 

0.5 

1.0 

0.8 
0.3 
0.8 
2.2 
2.1 
2.0 


>.2  o 


1610 

1655 
1875 
i860 
1630 
1630 
1675 
1625 
1650 
1205 
1 140 
1905 
1605 
1625 
1655 
465 
470 

145 
130 

385 
215 

290 
460 

1875 

3345 
2560 


FLOUR    AND    MEAL. 

As  illustrating  the  composition  of  a  common  vegetable  food  the  fol- 
lowing tests  may  be  made : 

Experiment.  Boil  a  small  amount  of  wheat  flour  with  Millon's  reagent.  The 
red  color  produced  shows  presence  of  proteins. 

Experiment.  Moisten  about  25  gm.  of  flour  with  water  and  work  it  into  a  dough. 
Then  hold  this  under  a  fine,  slow  stream  of  water  and  by  kneading  between  the 
fingers  slowly  work  out  a  portion  of  the  mass  as  a  thin  milky  liquid.  This  is 
largely  starch.  After  some  time  an  elastic  residue  is  left  insoluble  in  water.  This 
is  "  gluten "  and  is  the  chief  nitrogenous  element  of  the  flour,  which  has  been 
already  referred  to.     It  may  be  separated  into  several  constituents. 

Experiment.  To  about  5  gm.  of  flour  add  10  cc.  of  water,  shake  thoroughly  and 
allow  to  stand  until  a  nearly  clear  liquid  appears  above  a  white  sediment.  Filter 
the  liquid  and  test  for  sugar  by  the  Fehling  solution.  Boil  some  of  the  residue  with 
water  and  add  iodine  solution  as  a  test  for  starch. 

Experiment.  To  about  5  gm.  of  fine  corn  meal  in  a  test-tube  add  10  cc.  of  ether. 
Close  the  tube  with  the  thumb  and  shake  thoroughly.  Then  cork  and  allow  to  stand 
half  an  hour.  Shake  again  and  pour  the  mixture  on  a  small  filter,  collect  the 
ethereal  filtrate  in  a  shallow  dish  and  evaporate  it  by  immersion  in  warm  water. 
A  small  amount  of  fat  will  remain. 


THE    PROTEIN    SUBSTANCES.  95 

Action  of  Yeast  on  Flour.  The  following  experiment  is  intended  to  illustrate 
the  work  done  by  yeast  in  leavening  dough : 

Experiment.  Crumble  two  or  three  grams  of  compressed  yeast  into  15  cc.  of 
lukewarm  water  and  shake  or  stir  the  mixture  until  the  yeast  is  uniformly  dis- 
tributed. Then  stir  in  enough  flour  to  make  a  thick  cream  and  allow  to  stand  over 
night  at  room  temperature.  In  this  time  fermentation  of  the  small  amount  of  sugar 
in  the  flour  begins  and  the  "  sponge  "  swells  up  by  the  escape  of  bubbles  of  gas. 
At  this  stage  mix  in  uniformly  and  thoroughly  enough  flour  to  make  a  stiff  dough, 
using  for  the  purpose  perhaps  25  gm.  Put  the  dough  in  an  evaporating  dish,  keep 
it  for  an  hour  or  more  at  a  temperature  of  30°to  350  C.  and  observe  that  it  increases 
very  greatly  in  size,  from  the  continued  action  of  the  yeast  in  liberating  bubbles  of 
carbon  dioxide.  If  a  good  hot  air  oven  is  at  hand  the  experiment  is  completed  by 
baking  the  leavened  mass.  The  nature  of  the  yeast  fermentation  will  be  explained 
later. 

Milk.  In  milk  we  have  a  substance  in  which  all  the  essential  food 
elements  are  present.  The  average  composition  of  cow's  milk  is  given 
in  this  table. 

Water    87.4 

Fat     3.5 

Sugar 4.5 

Proteins    3.9 

Salts    07 

100.0 

Human  milk  contains  more  sugar  and  less  protein  than  the  milk  of 
the  cow.     Details  of  this  will  be  given  later. 


SECTION    II. 

FERMENTS   AND   DIGESTIVE   PROCESSES. 

CHAPTER   VI. 

ENZYMES   AND   OTHER   FERMENTS.    DIGESTION. 

In  the  course  of  time  the  conception  of  fermentation  has  undergone 
many  changes.  The  notion  was  first  associated  with  those  processes 
in  which  a  bubbling  or  boiling  condition  without  application  of  heat 
was  observed,  and  later,  as  the  most  familiar  kind  of  fermentation  was 
more  closely  studied,  this  phenomenon  was  found  to  be  due  to  the 
escape  of  gas.  This  escape  of  gas  came  finally  to  be  recognized  as  the 
essential  feature  of  fermentation  and  many  operations  bearing  no  rela- 
tion whatever  to  alcoholic  fermentation  were,  through  confusion  of 
ideas,  frequently  associated  with  it. 

The  real  fermentation,  which  follows  when  saccharine  juices  are 
exposed  to  the  air,  had  been  studied  in  a  way  from  the  remotest 
antiquity,  but  no  rational  attempts  at  an  explanation  of  the  process 
were  made  until  after  the  middle  of  the  seventeenth  century,  when  the 
relation  of  alcohol  and  the  gas  to  the  destruction  of  the  sugar  seems 
to  have  been  fully  recognized.  Several  other  reactions  were  asso- 
ciated with  the  alcoholic  fermentation;  in  the  leavening  of  bread  the 
production  of  a  gas  was  recognized,  and  it  was  noticed  that  in  the 
changes  going  on  in  the  animal  intestine  gases  were  also  liberated  fol- 
lowing the  digestion  of  foods.  Along  with  the  alcoholic  fermentation 
there  was  included  under  the  general  name  the  peculiar  change  which 
takes  place  when  the  wine  formed  from  saccharine  liquids  was  allowed 
to  stand  exposed  to  the  air.  To  be  sure  no  gas  was  formed  in  this 
action,  as  in  the  other,  but  something  in  common  was  recognized.  In 
both  cases  it  was  noticed  that  a  scum  formed  over  the  liquid  and  that 
a  small  amount  of  this  substance  was  capable  of  quickly  inciting  sim- 
ilar fermentation  in  more  saccharine  liquid  or  wine.  The  nature  of 
this  scum  became  in  time  the  subject  of  microscopic  investigation  (by 
Leuwenhock)  and  we  have  here  probably  the  beginning  of  our  real 
study  of  ferments.  It  was  in  1680  that  Leuwenhock  recognized  that 
this  scum  in  the  case  of  beer  yeast  consisted  of  minute  globules  with 
peculiar  properties;  the  full  value  of  this  discovery,  however,  was  not 

96 


ENZYMES    AND    OTHER    FERMENTS.  97 

generally  admitted  and  more  than  a  century  passed  before  any  great 
advance  was  made  by  others.  Lavoisier  toward  the  end  of  the  eight- 
eenth century  gave  the  first  explanation  of  the  chemistry  of  alcoholic 
fermentation,  as  he  was  able  to  point  out  the  relation  of  carbon  dioxide 
and  alcohol  to  the  parent  sugar.  But  the  cause  of  the  action  was  not 
much  discussed  and  just  what  the  function  of  the  cells  or  globules  of 
Leuwenhock  is  remained  obscure  until  the  time  of  Pasteur. 

Before  taking  up  the  important  work  of  Pasteur  something  must 
be  said  of  discoveries  in  other  directions.  The  older  conception  of 
fermentation  was  widened  by  the  addition  of  new  facts.  In  1780 
Scheele  isolated  lactic  acid  from  sour  milk  and  later  investigators 
began  to  look  for  the  agent  responsible  for  the  production  of  this  acid. 
About  1848  the  probable  nature  of  an  organism  which  appeared  to  be 
always  associated  with  lactic  fermentation  was  pointed  out  by  Blon- 
deau.  Various  formulas  were  given  for  the  production  of  lactic  acid 
in  quantity,  but  it  often  happened  that  the  final  product  was  an  entirely 
different  substance,  viz :  butyric  acid.  Butyric  fermentation  was 
therefore  added  to  the  list  of  these  peculiar  reactions,  and  various 
speculations  were  advanced  to  connect  the  different  phenomena.  Mean- 
while the  situation  became  still  further  complicated  by  the  gradual 
recognition  of  a  new  group  of  reactions  which  exhibited  many  of  the 
essential  features  of  the  alcoholic  and  acetic  fermentations,  and  which, 
therefore,  of  necessity  were  classed  as  ferment  reactions.  Several 
chemists  had  observed  the  peculiar  behavior  of  a  substance  produced  in 
germinated  barley;  this  substance  possessed  the  power  of  converting 
starch  into  a  sugar  which,  from  its  origin,  was  called  malt  sugar. 
Payen  and  Persoz,  in  1833,  succeeded  in  isolating  the  assumed  ferment 
from  the  sprouted  barley,  which  they  termed  diastase.  About  the 
same  time  it  was  recognized  that  saliva  contains  a  similar  starch- 
converting  agent  which  was  later  separated  and  called  salivary  diastase 
or  ptyalin.  The  seeds  of  the  bitter  almond  were  studied  by  several 
scientific  men  and  Liebig  and  Wohler  isolated  a  ferment  body  which 
they  termed  emulsin.  This  has  the  property  of  converting  the  gluco- 
side  called  amygdalin  into  glucose,  prussic  acid  and  oil  of  bitter 
almonds,  or  benzoic  aldehyde.  As  the  saliva  was  found  to  contain  a 
ferment  acting  on  starches,  so  the  gastric  juice  was  recognized  as 
active  through  the  presence  of  an  analogous  body  called  pepsin  which 
acts  on  proteins. 

Most  of  these  discoveries  were  made  before  1840.  The  ferments 
in  the  bitter  almond,  in  sprouted  barley,  in  the  saliva  and  in  the  gastric 
juice  were  all  found  to  be  soluble  in  water.  They  were  therefore 
8 


98  PHYSIOLOGICAL    CHEMISTRY. 

called  soluble  ferments  as  distinguished  from  the  yeasts  and  the  fer- 
ments of  acetic,  lactic  and  butyric  acids,  and  from  the  conditions  of 
their  action  Liebig  was  led  to  formulate  the  first  general  theory  of 
fermentation,  the  molecular  vibration  theory. 

THEORIES    OF    FERMENTATION. 

Liebig's  Theory.  Liebig  advanced  and  maintained  for  years  this 
view :  A  ferment  is  a  chemical  substance  whose  particles  or  molecules 
exist  in  a  peculiar  state  of  vibration,  and  in  contact  with  other  bodies 
this  ferment  is  able  to  set  up  similar  states  of  vibration  which  result 
in  the  breaking  down  of  the  bodies  mixed  with  the  ferment.  Fer- 
ments were  considered  along  with  bodies  undergoing  putrefaction,  and 
many  such  substances  were  supposed  to  be  able  to  bring  about  real 
fermentations.  According  to  a  somewhat  older  view  ferments  were 
said  to  act  by  their  mere  presence;  that  is,  they  exerted  what  was 
described  as  a  catalytic  action.  No  real  attempt,  however,  was  made 
to  define  more  closely  what  was  meant  by  this  catalytic  or  contact 
action. 

The  Theory  of  Pasteur.  The  real  nature  of  yeast  as  a  vegetable 
growth  had  finally  become  established.  With  this  admitted  Pasteur 
advanced  the  proposition  that  alcoholic  fermentation  is  a  consequence 
of  the  life  of  the  organism  in  contact  with  sugar  and  away  from  the 
air.  Alcohol  and  carbon  dioxide  are  products  of  the  yeast  cell  metabo- 
lism under  these  conditions.  The  cell,  according  to  the  Pasteur  view, 
must  be  furnished  with  a  proper  supply  of  oxygen  and  this,  under  the 
fermenting  conditions,  it  takes  from  the  sugar,  giving  off  carbon 
dioxide  as  an  oxidation  product  and  producing  alcohol  at  the  same 
time,  as  a  result  of  the  breaking  down  of  the  sugar  molecule.  Fer- 
mentation is  then  to  be  considered  from  a  purely  biological  standpoint 
with  alcohol  and  carbon  dioxide  as  excretory  and  respiratory  products 
respectively.  This  Pasteur  theory  soon  found  favor  with  the  majority 
of  scientific  men  and  gradually  supplanted  the  mechanical  notion  of 
Liebig  which  could  not  be  brought  into  accord  with  experience  in  other 
lines.  Although  the  Pasteur  view  that  the  yeast  produces  alcohol  only 
in  absence  of  free  oxygen  was  shown  to  be  incorrect  the  theory  com- 
mended itself  as  otherwise  satisfactory  and  tangible. 

Following  this  a  similar  explanation  was  offered  for  the  action 
taking  place  in  the  formation  of  acetic  acid,  lactic  acid  and  butyric 
acid.  Here  microorganisms  are  also  concerned.  These  live  on  cer- 
tain substances  and  produce  others  as  metabolic  excreta.  As  to  the 
mechanism  of  this  metabolism  we  know,  of  course,  nothing;  to  describe 


ENZYMES    AND    OTHER    FERMENTS.  99 

the  products  formed  as  excreta  is  perhaps  not  really  warranted  by  what 
is  actually  known.  It  must  be  remembered  then  that  the  term  is  used 
in  a  broad  and  general  sense  only  to  indicate  some  kind  of  a  metabolic 
product. 

The  work  of  Pasteur  gave  an  enormous  impetus  to  the  study  of  the 
common  fermentations,  but  it  was  evident  that  this  biological  expla- 
nation was  of  no  value  in  accounting  for  the  changes  produced  by  the 
active  agents  described  as  diastase,  pepsin,  emulsin,  etc.  These,  it  was 
pointed  out,  are  as  truly  "  ferments  "  as  are  yeast  and  the  mother  of 
vinegar.  To  avoid  confusion  it  became  customary  to  speak  of  the 
organised  and  unorganised  ferments,  or  the  insoluble  and  soluble  fer- 
ments. The  term  ensyme  was  later  applied  to  these  soluble  unorgan- 
ized agents  of  change,  but  this  new  expression  did  nothing  toward 
explaining  the  difficulty  or  toward  relating  the  two  classes  of  ferments. 

Certain  scientists  from  the  start,  however,  refused  to  admit  any 
fundamental  difference  between  the  work  of  the  yeast  ferment  on  the 
one  hand  and  that  of  bodies  like  diastase  on  the  other.  Even  after 
the  biological  theory  of  Pasteur  had  become  current  Berthelot,  Hoppe- 
Seyler  and  other  chemists  of  prominence  maintained  that  the  living 
cell  ferments  are  active  because  they  secrete  soluble  or  enzymic  bodies. 
In  the  one  case  the  actual  "  fermentation  "  takes  place  within  the  cell, 
as  appeared  to  be  the  fact  with  yeast;  in  other  cases  enzymes  are  pro- 
duced by  cells  and  thrown  off  to  do  their  work  elsewhere.  This  is 
true,  for  example,  in  the  stomach  where  certain  groups  of  cells  produce 
the  active  ferment  pepsin  which,  however,  does  its  work  of  dissolving 
coagulated  protein,  or  digesting  it,  outside  the  cells  themselves.  In 
germinating  barley  the  living  cells  secrete  diastase  which  may  be 
leached  out  and  used  to  digest  starch  of  other  grains.  If  not  leached 
out  the  diastase  gradually  digests  the  starch  of  the  barley  kernel  itself, 
unless  the  action  be  checked  by  heat  or  other  means. 

The  Work  of  Buchner.  In  principle,  therefore,  the  two  kinds  of 
ferment  action  were  held  to  be  alike,  but  although  many  attempts  were 
made  no  chemist  succeeded  in  isolating  the  assumed  enzyme  from  the 
active  cells.  Repeated  failure  in  this  direction  only  served  to  strengthen 
the  belief  of  the  advocates  of  the  vital  theory  according  to  which  alco- 
holic and  similar  fermentations  by  fungi  are  processes  which  cannot 
be  thought  of  dissociated  from  the  function  of  life  itself.  But  finally 
the  problem  was  solved  by  the  German  chemist  Buchner,  who  in  1897 
succeeded  in  isolating  the  active  enzyme  from  yeast  cells  and  in  quan- 
tity too.  This  enzyme  he  called  symase;  it  was  found  to  be  as  active 
as  the  yeast  itself  and  to  do  all  that  could  be  expected  of  yeast.     It  has 


IOO  PHYSIOLOGICAL    CHEMISTRY. 

since  been  produced  on  the  commercial  scale  in  the  endeavor  to  sup- 
plant the  use  of  yeast  in  practice.  More  recently  it  has  been  shown 
that  other  ferment  cells  secrete  enzymes  and  it  is  possible  that  all  the 
so-called  organized  ferments  work  in  this  way;  but  the  isolation  of  the 
soluble  active  principle  seems  to  be  very  difficult  in  most  cases. 

All  this,  however,  affords  us  no  real  insight  into  the  nature  of  the 
ferment  process.  We  have  as  yet  no  satisfactory  theory  as  to  how 
these  active  chemical  principles  behave  in  the  breaking  down  of  other 
organic  substances.  It  has  not  been  found  possible  to  prepare  any 
enzyme  in  a  condition  of  even  approximate  purity  and  all  analyses 
made  of  such  substances  are  doubtless  wide  of  the  truth.  These 
analyses  appear  to  show  that  the  enzymes  are  of  protein  character,  but 
the  impurities  in  the  products  analyzed  may  be  responsible  for  this 
indication.  With  this  lack  of  knowledge  regarding  the  chemical  com- 
position of  the  ferments  it  is  naturally  impossible  to  offer  a  chemical 
explanation  of  how  they  act.  It  is  the  effects  only  that  we  are  familiar 
with  and  all  our  classifications  are  practically  based  on  what  the  fer- 
ments can  do  rather  than  on  what  they  are.  It  is  known  that  all 
ferments  are  destroyed  by  heat  and  by  the  action  of  even  rather  weak 
acids  and  alkalies.  In  this  they  resemble  the  cells  that  produce  them. 
While  the  true  ferments  or  enzymes  are  apparently  complex  chemical 
substances  their  formation  is  due  in  every  case,  as  least  it  so  appears, 
to  cell  action.  They  are  organic,  but  not  organized;  yet  they  possess 
many  of  the  properties  of  organized  bodies.  On  the  other  hand  cer- 
tain finely  divided  metals,  especially  colloidal  platinum,  bring  about  a 
number  of  reactions  which  were  long  supposed  to  be  characteristic  of 
the  true  ferments;  these  reactions  are  further  modified  or  suspended 
by  the  same  substances  which  modify  or  suspend  the  ferment  actions 
in  question.  Based  on  this  behavior  it  has  been  attempted  to  relate 
the  true  ferment  action  to  the  "  catalytic  "  action  of  the  "  inorganic  " 
ferments.  All  the  ferments  seem  to  have  the  power  of  decomposing 
hydrogen  peroxide  in  quantity  or  catalytically,  and  this  property  has 
been  considered  as  perfectly  typical  or  characteristic.  The  addition  of 
a  number  of  mineral  substances  interferes  with  this  catalytic  decom- 
position ;  in  this  respect  the  action  of  prussic  acid  is  remarkably  ener- 
getic. It  has  been  found  that  a  minute  trace  of  colloidal  platinum  in 
dilute  solution  decomposes  a  greatly  excessive  amount  of  the  peroxide, 
and  further  that  extremely  dilute  prussic  acid  or  corrosive  sublimate 
checks  the  reaction  here  just  as  with  the  true  ferment.  These  analogy 
reactions  are  very  suggestive,  even  if  they  do  not  explain. 

Considering  the  enzymes  as  catalytic  agents  it  is  to  be  noted  that  their 


ENZYMES    AND    OTHER    FERMENTS.  IOI 

distinctive  function  is,  therefore,  to  hasten  certain  changes  which 
would  take  place  without  them,  but,  in  many  cases,  with  extreme  slow- 
ness. They  have  not  only  the  power  of  hastening  or  effecting  decom- 
positions, as  in  the  alteration  of  sugars  or  starches,  but  also  of  effecting 
many  syntheses.  For  example,  maltase  has  the  power  of  bringing 
about  the  conversion  of  glucose  into  isomaltose.  In  some  cases  the 
enzymes  act  to  retard,  in  place  of  hastening  reactions,  and  in  a  great 
number  of  instances  they  seem  to  act  as  aids  to  other  catalyzers.  In 
this  sense  they  are  spoken  of  as  co-enzymes,  kinases  or  activators. 

Enzymes  consist  of  very  large  molecules  which  are  usually  unable 
to  pass  through  animal  membranes  or  fine  porcelain  filters.  In  cases 
where  they  can  be  so  filtered  the  rate  of  passage  is  very  slow.  In  addi- 
tion to  large  size  and  probably  complex  structure  the  enzymes  seem 
to  possess  a  certain  sort  of  specificity.  That  is,  their  activity  is  exerted 
in  certain  directions,  or  on  certain  compounds  only.  The  enzyme 
which  aids  the  conversion  of  starch  into  sugar  is  inactive  as  far  as  the 
digestion  of  albumin  is  concerned,  although  the  reactions  have  much 
in  common.  But  the  specific  behavior  does  not  end  here.  Certain 
enzymes  will  effect  a  decomposition  in  one  form  of  a  glucoside,  but 
not  in  its  optical  isomer,  and  in  a  large  number  of  reactions  it  has  been 
found  that  pancreatic  extracts  will  hydrolyze  certain  artificial  poly- 
peptides, but  not  their  isomers,  or  related  bodies.  Such  behavior  points 
to  a  peculiar  chemical  structure  on  the  part  of  the  enzyme  which  must 
bear  some  relation  to  the  structure  of  the  thing  acted  upon,  or  the 
"  substratum,"  as  it  is  frequently  called.  Following  out  this  idea  there 
has  been  no  little  speculation  as  to  the  manner  in  which  the  enzymes 
hasten  reactions.  It  appears  that  the  enzyme,  through  its  structural 
configuration,  unites  with  the  substratum  in  such  a  way  as  to  produce 
a  new  compound  which  yields  the  same  end  products  as  the  substratum 
alone  would  yield,  but  much  more  rapidly.  In  the  decomposition  to 
furnish  the  end  products  the  enzyme  group  is  liberated  to  combine  with 
a  new  portion  of  substratum,  and  so  on,  until  the  reaction  is  complete, 
or  has  reached  a  condition  of  equilibrium.  It  has  been  suggested  that 
enzymes  act  as  very  weak  acids  or  weak  bases,  or  that  they  combine 
both  properties  as  do  the  amino  acids,  and  through  this  behavior  they 
are  able  to  unite  with  corresponding  groups  of  the  substratum.  There 
is  evidence  that  in  a  number  of  such  combinations  studied  the  reaction 
follows  the  mass  action  laws  with  a  fair  degree  of  closeness. 


102  PHYSIOLOGICAL    CHEMISTRY. 

CLASSIFICATION  OF  THE  FERMENTS. 

With  our  present  knowledge  of  ferments  they  are  most  satisfactorily 
classified  according  to  the  character  of  the  .decompositions  they  effect. 
Two  distinct  kinds  of  action  are  easily  recognized.  Many  ferment 
changes  are  clearly  hydrolytic ;  that  is,  the  reaction  follows  through  the 
addition  of  a  molecule  or  more  of  water  to  one  substance,  causing  it 
to  break  up  into  smaller  groups.  In  other  cases  the  reaction  appears 
to  be  in  the  nature  of  an  oxidation  process  in  which  the  ferment  causes 
or  brings  about  the  addition  of  oxygen  to  convert  one  substance  into 
another.  Some  authors  limit  the  true  ferment  reactions  to  changes 
which  may  be  referred  to  one  or  the  other  of  these  heads.  But  there 
are  a  great  many  decompositions  which,  while  they  may  not  be  so 
clearly  defined  as  those  just  mentioned,  must  still  be  looked  upon  as 
of  ferment  origin.  These  are  produced  by  bacteria,  and  in  all  proba- 
bility by  the  enzymes  secreted  by  bacteria.  It  will  be  well  therefore 
to  add  a  third  general  group  to  make  the  classification  complete. 

KINDS  OF  FERMENT  REACTIONS. 

We  may  make  three  general  divisions  of  the  ferment  changes,  as 
follows : 

A.  Hydrolytic  Reactions. 

B.  Oxidation  Reactions. 

C.  Bacterial  Decompositions. 

A  brief  discussion  of  the  more  important  changes  coming  under 
each  one  of  these  heads  will  now  follow. 

A.     HYDROLYTIC    REACTIONS. 

The  most  important  of  our  ferment  reactions,  with  one  exception, 
perhaps,  and  at  the  same  time  the  most  thoroughly  studied,  the  changes 
involving  hydrolysis  have  long  claimed  the  attention  of  chemists.  The 
true  nature  of  some  of  these  reactions  is  easily  recognized  and  the 
earlier  workers  in  this  field  were  able  to  compare  the  behavior  of  the 
enzymes  in  question  with  that  of  dilute  hydrochloric  or  sulphuric 
acid.  In  other  very  important  cases  this  analogy  is  far  less  readily 
pointed  out  and  it  remained  for  recent  workers  to  satisfactorily  estab- 
lish the  true  relations.  When  malt  digests  starch  or  when  certain 
enzymes  convert  the  malt  sugar  formed  into  glucose  the  general  nature 
of  the  changes,  as  requiring  the  addition  of  water,  may  be  shown 
without  difficulty.  But  with  the  behavior  of  pepsin  in  digesting  pro- 
tein we  have  more  difficulty.  Here  the  reaction  is  not  so  easily  fol- 
lowed, and  the  quantitative  relations  between  the  original  substances 


ENZYMES    AND    OTHER    FERMENTS.  103 

and  the  products  formed  are  more  complicated  than  is  the  case  with 
the  carbohydrate  decompositions.  However,  these  reactions  likewise 
have  been  shown  to  involve  true  cases  of  water  addition  and  therefore 
may  be  properly  grouped  with  the  carbohydrate  reactions  as  hydrolytic. 
This  hydrolytic  ferment  activity  is  exhibited  mainly  in  the  following 
directions : 

1.  In  the  modification  of  carbohydrates  as  illustrated  by  the  sac- 
charification  of  starch  and  further  changes  in  the  sugar  thus  formed, 
and  in  other  sugars. 

2.  In  the  breaking  down  of  glucosides. 

3.  In  the  splitting  of  fats. 

4.  In  the  digestion  of  proteins. 

5.  In  the  so-called  fermentation  of  urea. 

Some  of  these  reactions  may  be  represented  by  definite  equations. 
In  general  they  correspond  to  the  changes  produced  in  the  same  sub- 
stances by  weak  acids  with  some  variations  in  the  details.  The  salient 
points  will  be  indicated  here,  leaving  in  most  cases  the  fuller  discussion 
for  following  chapters  which  deal  with  the  details  of  the  digestion 
phenomena. 

CHANGES    IN    CARBOHYDRATES. 
Amylase  or  Diastase.     Certain  enzymes  convert  starch  paste  into 
malt  sugar  by  a  reaction  which  is  indicated  by  the  equation : 

(  CjjH^oO^)  n  +  (HoO)ra=  (C^H^OiOn 

The  enzymes  here  active  are  usually  described  as  diastases  or  amylases, 
the  terms  being  employed  in  the  plural,  since  the  action  is  not  confined 
to  a  single  substance.  Of  these  two  terms  the  word  diastase  is  fre- 
quently employed  in  the  broad  sense  to  include  all  the  enzymes  which 
act  on  the  starches  and  sugars  formed  from  them,  while  the  term 
amylase  is  employed  to  describe  the  enzyme  which  changes  starch  into 
malt  sugar.  In  this  sense  it  will  be  used  here.  In  nature  ferments 
of  this  character  are  very  widely  distributed  and  serve  very  important 
functions.  They  are  active  in  the  changes  going  on  in  the  vegetable 
kingdom  during  the  growth  of  plants  and  the  ripening  of  fruits,  as 
well  as  in  the  germination  of  seeds.  On  the  commercial  scale  malt 
represents  the  best  known  diastase-containing  substance.  In  the  ani- 
mal body  similar  substances  are  found  in  the  saliva  and  in  the  pan- 
creatic secretion.  The  first  of  these  is  called  salivary  diastase  or 
ptyalin  and  the  second  pancreatic  diastase  or  amylopsin. 

These  diastases  have  never  been  secured  in  anything  like  pure  con- 
dition.    Very  active  solutions  which  digest  starch   quickly  may  be 


104  PHYSIOLOGICAL    CHEMISTRY. 

obtained  by  extracting  ground  malt  with  water,  which  will  be  illus- 
trated later.  These  solutions  may  be  concentrated  at  a  moderate  tem- 
perature, but  the  activity  of  the  enzyme  is  destroyed  by  heat.  A 
stronger  product  may  be  secured  by  extracting  with  20  per  cent  alcohol 
and  precipitating  the  solution  so  obtained  by  absolute  alcohol.  This 
precipitate  in  turn  may  be  redissolved  in  water  and  precipitated  again 
with  strong  alcohol  or  with  ammonium  sulphate,  to  secure  a  purer  and 
more  active  product. 

Besides  the  well-known  ferments  in  malt,  in  the  saliva  and  in  the 
pancreatic  secretion  the  following  may  be  mentioned  here.  By  many 
authors  the  active  substance  in  the  liver  which  converts  glycogen  into 
glucose  is  supposed  to  be  a  form  of  diastase.  Others  hold  the  conver- 
sion of  sugar  into  glycogen  and  the  subsequent  and  gradual  formation 
of  sugar  from  glycogen  to  be  specific  vital  functions  performed  by  the 
liver  cells.  The  name  cellulase  or  cytase  is  given  to  a  ferment  body 
which  is  found  in  many  vegetable  substances  and  which  has  the  power 
of  converting  cellulose  into  sugar.  Inulase  is  the  enzyme  which  acts 
on  the  peculiar  starch  known  as  inulin  found  in  many  vegetable  sub- 
stances, converting  it  into  fructose.  Inulase  does  not  appear  to  act  on 
ordinary  starch  and  on  the  other  hand,  malt  diastase  is  not  able  to 
convert  inulin  into  sugar.  Pectinase  is  another  little  known  vegetable 
enzyme  which  converts  the  so-called  pectin  jelly  substances  into  a 
reducing  sugar.  The  original  pectose  in  the  seed  or  fruit  is  first 
changed  into  pectin  by  a  kind  of  coagulating  enzyme  called  pectase. 
The  true  behavior  of  these  bodies  is  not  yet  fully  known. 

Maltase  or  Glucase.  The  sugar  formed  by  amylase  from  starch 
is  known  as  maltose.  This  is  a  primary  product  and  may  readily  be 
further  converted  into  glucose  by  another  enzyme  occurring  in  malt 
and  properly  known  as  maltase.  The  nomenclature  of  these  enzymes 
is  unfortunately  somewhat  confused.  An  effort  has  been  made  to 
name  them  systematically,  using  in  each  case  the  name  of  the  carbo- 
hydrate or  other  body  on  which  the  enzyme  acts,  as  the  first  part  of 
the  descriptive  term,  to  be  followed  by  the  suffix  ase.  Thus  amylase 
refers  to  the  enzyme  acting  on  amy  lose  or  starch  and  maltase  to  the 
enzyme  which  acts  on  maltose  or  malt  sugar.  But  many  authors  do 
not  follow  this  system  consistently;  hence  we  have  as  describing  the 
same  ferment  the  term  glucase  in  use,  since  glucose  is  the  product 
formed.  It  is  preferable  to  employ  the  first  designation  or  maltase. 
This  enzyme  belongs  to  the  class  of  so-called  inverting  ferments  which 
convert  disaccharides  into  monosaccharides.  In  this  special  case  malt 
sugar  yields  glucose : 


ENZYMES    AND    OTHER    FERMENTS.  105 

CuHaA,  +  H20  =  2C6H12Oc. 

This  maltase  is  found  not  only  in  malt  extract,  but  in  various  yeasts 
and  elsewhere  in  the  vegetable  kingdom.  It  is  also  present  in  saliva, 
but  in  small  amount,  in  the  pancreas,  the  liver  and  in  the  blood.  The 
formation  of  glucose  in  most  cases  is  probably  a  secondary  reaction, 
maltose  being  formed  first  as  the  primary  product.  The  general  im- 
portance of  this  reaction  will  be  pointed  out  later,  as  it  plays  a  very 
essential  part  in  the  digestion  of  the  carbohydrate  foods. 

Lactase.  In  analogy  with  the  conversion  of  malt  sugar  into  glucose 
we  have  the  conversion  of  its  isomer,  lactose  or  milk  sugar,  into  two 
monosaccharide  groups.  This  is  accomplished  by  the  ferment  called 
lactase  which  is  found  in  several  kinds  of  yeast,  and  which  appears  to 
be  distinct  from  the  maltase  just  described.  The  change  of  milk 
sugar  is  represented  by  this  reaction : 

CaH^O*  +  H20  =  CH^O,  +  CH^O.. 

Glucose         Galactose 

Lactose  and  glucose  have  nearly  the  same  specific  rotation,  [oc]z)=52.5° 
for  the  first  and  53 °  for  the  second,  while  for  the  galactose  it  is  about 
83  °.  The  inversion  may  be  readily  followed  by  the  polariscope, 
therefore. 

As  to  the  distribution  of  this  enzyme  in  nature  there  is  still  some 
dispute.  According  to  some  authors  lactase  is  not  present  in  the  gas- 
tric juice  or  in  the  pancreatic  secretion,  but  other  investigators  have 
reported  finding  it  in  both  secretions.  It  was  formerly  held  that  the 
disappearance  of  milk  sugar  in  the  body  is  due  largely  to  bacterial 
actions,  as  some  of  these  organisms  are  able  to  secrete  an  enzyme 
which  acts  on  the  sugar. 

Invertase  or  Sucrase.  One  of  the  most  common  and  important  of 
these  enzymic  reactions  is  the  inversion  of  cane  sugar,  forming  glucose 
and  fructose. 

C12H22On  +  H20  =  C6H1208  +  C.HuO.. 

Glucose         Fructose 

The  name  invertin  or  invertase  has  been  given  to  the  enzyme  which 
accomplishes  this,  but  sucrase  would  be  in  better  accord  with  the  gen- 
eral nomenclature.  The  presence  of  this  inverting  ferment  in  many 
kinds  of  yeast  has  been  long  known.  The  yeast  cell  alone  is  not  able 
to  convert  cane  sugar  into  alcohol  and  carbon  dioxide;  an  inversion 
must  first  be  brought  about  in  some  manner.  In  old  yeast  or  in  yeast 
in  which  the  cell  has  been  destroyed  by  heat  or  by  mechanical  means 
the  inverting  enzyme  seems  to  be  present  in  greatest  abundance. 
Invertase  is   found  in  various  animal  secretions,  especially  in  the 


106  PHYSIOLOGICAL    CHEMISTRY. 

intestinal  juice.  The  inverting  power  of  this  secretion  is  marked, 
while  with  the  pancreatic  secretion  the  inverting  power  is  much  less 
pronounced.  In  the  gastric  juice  the  inverting  enzyme  is  said  to  be 
present  in  some  amount  and  is  sufficient  to  change  part  of  the  cane 
sugar  of  the  food  independently  of  the  acid  likewise  present. 

The  blood  does  not  appear  to  contain  this  invertase,  since  a  solution 
of  cane  sugar  injected  into  the  veins  is  eliminated  later  by  the  kidneys 
unchanged.  If  injected  into  the  portal  vein,  and  thus  made  to  pass 
the  liver,  inversion  takes  place  rapidly,  as  that  organ  possesses  the 
enzyme  in  quantity. 

Many  of  the  higher  as  well  as  the  lower  plant  organisms  contain 
invertase,  which  accounts  for  the  change  of  the  cane  sugar  into  invert 
sugar  in  certain  cases.  In  general  this  reaction  may  be  easily  fol- 
lowed by  the  polariscope,  as  the  strong  dextro-rotation  of  cane  sugar 
gives  place  to  the  levo-rotation  of  invert  sugar.  It  is  possible  to  make 
a  fairly  pure  invertase  solution  from  some  kinds  of  yeast,  and  such  a 
solution  has  certain  practical  applications  in  analytical  investigations. 
By  extracting  yeast  with  thymolized  water  a  solution  is  obtained  which 
rapidly  inverts  cane  sugar,  but  which  is  practically  without  action  on 
malt  sugar  or  milk  sugar  and  which,  at  the  same  time,  will  not  induce 
alcoholic  fermentation.  This  property  of  the  yeast  extract  is  made 
use  of  in  the  determination  of  cane  sugar  in  presence  of  the  others  just 
mentioned. 

GLUCOSIDE   REACTIONS. 

For  our  purpose  it  will  not  be  necessary  to  go  into  many  details  here. 
A  few  decompositions  only  need  be  mentioned  by  way  of  illustration. 
The  glucosides  are  peculiar  compounds  which  may  be  looked  upon  as 
more  or  less  complex  ethers  of  glucose.  They  are  decomposed  in 
various  cleavage  processes,  with  the  separation,  usually,  of  glucose  as 
one  of  the  constituent  products. 

Emulsin.  The  best  known  reaction  in  this  group  is  that  which 
takes  place  spontaneously  in  the  crushed  bitter  almond.  Along  with 
other  substances  this  kernel  contains  a  characteristic  nitrogenous  gluco- 
side  known  as  amygdalin  and  the  enzyme  called  emulsin.  In  presence 
of  water  the  amygdalin  breaks  up  in  this  way : 

CJH^NOa  +  2H20  =  aQH^Oe  +  HCN  +  CGHBCHO, 

that  is,  glucose,  hydrocyanic  acid  and  benzoic  aldehyde  are  formed. 
In  the  uncrushed  dry  almond  this  reaction  does  not  take  place  because 
the  enzyme  and  glucoside  are  not  in  direct  contact,  but  are  contained 
in  different  cells.  The  same  result  is  accomplished  by  distillation  of 
the  bitter  almond  with  dilute  acids. 


ENZYMES    AND    OTHER    FERMENTS.  107 

Similar  reactions  are  observed  with  salicin, 

C13H1S07  +  H;0  =  C.HuO,  +  C6H4.OH.CH2OH, 

Saliginin 

and  with  coniferin,  arbutin  and  other  glucosides.  A  related  ferment, 
known  as  myrosin,  converts  the  potassium  myronate  found  in  black 
mustard  into  allyl  mustard  oil,  C3H5NCS,  glucose,  and  potassium  acid 
sulphate. 

THE    SPLITTING   OF    FATS. 
The  general  reactions  of  fats  have  been  already  referred  to  and  it 
has  been  shown  that  in  general  they  may  be  broken  up  by  the  action 
of  water  in  the  form  of  superheated  steam : 

C3H5(CnH2„.102)s  +  3H20  =  C3H503H3  +  3^CnU2n^02. 

The  action  of  the  pancreas  in  the  emulsification  of  fats  was  recognized 
as  early  as  1834  and  in  seeking  for  the  cause  of  this  it  was  finally 
found  to  depend  on  a  ferment  reaction,  and  subsequent  soap  formation. 
Lipase  or  Steapsin.  The  active  principle  in  the  pancreas  which 
accomplishes  this  fat  splitting  was  first  called  steapsin  and  afterwards 
lipase.  The  details  of  its  behavior  in  the  digestion  of  fats  will  be 
explained  in  a  following  chapter.  Besides  its  constant  occurrence  in 
the  pancreas,  it  has  been  found  in  the  blood,  the  liver  and  the  kidney. 
More  recently  the  existence  of  lipase  in  many  vegetable  substances  has 
been  observed  and  thoroughly  studied.  It  has  been  found  that  it 
hydrolyzes  some  of  the  simpler  ethereal  salts  very  readily  and  on  this 
behavior  is  based  a  method  of  recognition  of  convenient  application. 
Of  these  ethereal  salts  ethyl  butyrate  is  possibly  the  best,  as  it  suffers 
but  very  slight  change  by  the  action  of  water  alone  at  ordinary  tem- 
peratures. The  fat-splitting  power,  or  enzyme  strength,  of  various 
extracts  may  be  compared  by  noting  the  amount  of  the  ethyl  butyrate 
decomposed  in  a  given  time  under  the  influence  of  the  extract.  The 
extent  of  hydrolysis  of  the  ethereal  salt  is  determined  by  titrating  the 
butyric  acid  liberated  with  dilute  alkali. 

PROTEOLYTIC    REACTIONS. 

While  it  is  not  possible  to  write  equations  illustrating  accurately  the 
absorption  of  water  in  the  digestion  of  proteins,  as  may  be  done  for 
the  carbohydrates  and  the  fats,  yet  there  is  abundant  evidence  to  show 
that  water  addition  is  in  most  cases  the  characteristic  preliminary 
change  here  also.  The  action  of  superheated  steam  has  been  referred 
to  in  a  former  chapter,  but  at  the  ordinary  temperature  certain  pro- 
teolytic changes  take  place  which  are  the  results  of  enzyme  action.     At 


108  PHYSIOLOGICAL    CHEMISTRY. 

least  three  of  these  changes  have  been  thoroughly  studied,  and  are  of 
great  importance  in  the  digestion  of  foods. 

Rennet  or  Rennin.  It  has  long  been  known  that  a  certain  product 
found  in  the  stomachs  of  young  animals  and  especially  in  the  calf's 
stomach,  has  the  power  of  clotting  milk  rapidly,  which  property  has 
been  applied  in  the  manufacture  of  cheese.  The  same  substance  is 
found  also  in  the  pancreas,  and  the  same  or  a  quite  similar  enzyme 
in  a  number  of  plants.  In  fact,  this  curdling  or  clotting  ferment,  like 
others  already  described,  is  quite  widely  distributed  in  nature.  As 
occurring  in  the  stomach  it  is  mixed  with  another  ferment,  which  will 
be  described  below,  known  as  pepsin.  The  two  substances  are  appar- 
ently quite  distinct  from  each  other  and  may  be  more  or  less  perfectly 
separated.  Some  chemists  are,  however,  inclined  to  consider  them  as 
essentially  similar. 

Rennet  acts  on  the  protein  substance  casein,  throwing  it  into  a  coag- 
ulated or  clotted  form.  The  chemistry  of  the  reaction  is  obscure  and 
not  thoroughly  worked  out.  The  essentials  of  what  is  known  about 
it  will  be  given  later.  It  is  possible  to  obtain  an  active  extract  from 
the  stomach  of  the  calf  or  young  pig  which  may  be  kept  indefinitely 
and  used  for  cheese  making  or  other  purposes.  Formerly  in  the  cheese 
industry  small  fragments  of  the  dried  calf's  stomach,  preserved  for  the 
purpose,  were  mixed  with  the  milk  and  stirred  about  to  induce  coagu- 
lation. At  the  present  time  a  liquid  extract  is  made  on  the  commercial 
scale  by  the  action  of  an  appropriate  solvent  on  the  cleaned  stomach. 
Glycerol  may  be  used,  or  water  plus  a  small  amount  of  salicylic  acid 
to  prevent  putrefaction.  In  some  European  countries  certain  plants 
have  been  employed  in  the  place  of  animal  rennet  in  the  cheese  indus- 
try. Rennet  works  well  in  an  acid  medium  and  is  easily  destroyed  by 
alkalies. 

Pepsin.  The  best  known  and  most  thoroughly  studied  of  the  pro- 
teolytic enzymes  is  pepsin  which  has  the  power  of  digesting  coagulated 
albumin  in  an  acid  medium.  It  may  be  obtained  best  from  the  mucous 
membrane  of  the  hog's  stomach  by  extraction  with  acidulated  water 
or  glycerol.  In  the  stomach  it  appears  to  exist  as  a  propepsin  or 
zymogen,  in  which  condition  it  is  known  as  pepsinogen.  The  action 
of  acid  converts  this  into  the  true  ferment.  Pepsin  is  very  sensitive 
to  the  action  of  alkalies  which  even  in  weak  solution  destroy  it  or 
materially  lessen  its  power  of  dissolving  protein.  In  presence  of  weak 
acids,  preferably  hydrochloric  acid  of  o.i  to  0.2  per  cent  strength,  it 
forms  from  the  native  or  coagulated  proteins  the  derived  products 
known  as  albumoses  and  peptones.     This  change  is  unquestionably 


ENZYMES    AND    OTHER    FERMENTS.  IO9 

associated  with  the  addition  of  a  number  of  molecules  of  water  to  the 
original  protein  group. 

Commercial  "  pepsin  "  appears  in  commerce  in  the  form  of  powder 
or  scales.  The  latter  are  obtained  by  drying  the  extract  from  the 
glands  of  the  stomach  on  glass  plates.  The  product  is  far  from  pure, 
as  it  contains  a  large  excess  of  other  extractives.  Yet,  as  now  made, 
one  part  by  weight  of  the  scale  or  powder  is  capable  of  digesting  or 
rendering  soluble  two  to  four  thousand  parts  of  coagulated  albumin  in 
the  form  of  hard-boiled  eggs.  In  an  experimental  way  products  of 
enormously  greater  activity  have  been  prepared;  it  is  said  that  one 
part  of  a  dry  pepsin  may  be  made  to  dissolve  three  hundred  thousand 
parts  of  coagulated  egg  albumin.  The  relative  strengths  of  pepsin 
products  are  always  compared  by  noting  the  amount  of  egg  albumin 
or  washed  fibrin  which  they  will  digest  in  an  acid  medium  of  definite 
concentration. 

Pepsin,  like  most  of  the  enzymes,  is  precipitated  by  alcohol.  In 
aqueous  solution  with  a  little  acid  it  is  most  active  at  about  400  C, 
and  loses  its  power  at  about  560.  In  the  dry  condition  it  withstands 
perfectly  a  much  higher  temperature.  While  hydrochloric  acid  is 
usually  employed  as  an  aid  to  pepsin  digestion,  other  acids  may  be  used 
with  equally  good  results.  Oxalic  acid,  lactic  acid  and  formic  acid 
work  well,  but  the  action  with  acetic  and  propionic  acids  is  weak.  In 
presence  of  alkalies  there  is  no  activity  and  certain  salts  also  interfere 
with  the  digestive  power. 

Through  fractional  precipitations  and  by  other  means  many  attempts 
have  been  made  to  obtain  a  "  pure  "  pepsin.  The  strongest,  that  is,  the 
most  active,  products  so  secured  have  been  analyzed.  The  results  do 
not  differ  greatly  from  those  found  on  analyzing  the  proteins,  yet  with 
some  of  these  products  it  is  not  possible  to  obtain  the  ordinary  protein 
reactions.  We  have  no  clew  to  the  real  composition  of  the  substance. 
It  is  not  at  all  diffusible  through  parchment  and  must  have  a  high 
molecular  weight. 

It  is  stated  above  that  pepsin  and  rennin  are  possibly  identical  substances.  The 
view  commonly  held  by  the  majority  of  chemists  and  physiologists  has  been  that 
they  are  distinct  ferments  produced  possibly  by  different  regions  of  the  stomach,  but 
in  late  years  a  mass  of  evidence  has  been  accumulating  which  appears  to  throw 
doubt  on  this  notion  and  suggest  the  perfect  identity  of  the  two  proteolytic  enzymes. 
The  chemists  of  the  Pawlow  school  have  been  particularly  active  in  advancing  this 
theory.  According  to  them  an  extract  which  shows  the  digesting  power  will  also 
show  the  milk  curdling  action.  If  it  is  strong  in  the  one  case  it  will  be  found 
strong  in  the  other  if  the  conditions  are  made  right.  This  amounts  to  saying  that 
the  same  enzyme  does  the  two  kinds  of  work,  but  the  conditions  of  reaction,  con- 
centration, salt  content,  etc.,  must  be  different  in  each  case.     A  commercial  rennet, 


IIO  PHYSIOLOGICAL    CHEMISTRY. 

for  example,  if  largely  diluted  with  0.2  per  cent  hydrochloric  acid,  will  show  a  strong 
proteolytic  reaction,  while  without  such  dilution  it  may  appear  quite  inactive.  It  is 
held  further  that  the  milk  curdling  ferment  of  the  pancreatic  extract  is  probably 
identical  with  the  trypsin  to  be  now  described. 

Trypsin.  One  of  the  most  active  and  important  of  the  body  fer- 
ments is  the  substance  which  occurs  in  the  pancreas  and  known  as 
trypsin.  It  may  be  extracted  from  the  minced  organ  in  a  variety  of 
ways  and  in  crude  form  is  a  commercial  product.  In  its  action  it  bears 
some  resemblance  to  pepsin,  but  works  under  different  conditions. 
While  pepsin  digests  protein  compounds  in  dilute  acid  medium  trypsin 
is  most  active  in  presence  of  weak  alkali,  preferably  sodium  carbonate. 
Action  may  be  observed,  however,  in  neutral  solution  and  even  in  pres- 
ence of  a  trace  of  acid.  In  its  hydrolysis  of  proteins  trypsin  goes 
farther  than  pepsin.  The  action  of  the  latter,  under  ordinary  condi- 
tions, ends  with  the  production  of  albumoses  and  peptones,  while  the 
pancreatic  enzyme  carries  the  splitting  process  to  the  extent  of  pro- 
ducing a  number  of  comparatively  simple  amino  acids,  and  the  hexone 
bases.  In  this  respect  the  behavior  of  the  trypsin  is  comparable  with 
that  of  weak  sulphuric  acid  when  heated  with  the  protein  bodies.  As 
already  pointed  out  in  a  former  chapter  this  acid  resolves  the  proteins 
into  complexes  which  may  be  considered  as  the  constituent  groups  of 
the  large  molecule.  The  trypsin  digestion  may  be  carried  far  enough 
to  leave  products  which  fail  to  show  more  than  a  very  faint  biuret 
reaction.  This  reaction,  it  will  be  remembered,  persists  as  long  as 
anything  having  the  characteristics  of  the  original  protein  substance 
remains.  From  all  this  it  is  evident  that  the  trypsin  is  a  hydrolyzing 
agent  of  marked  power. 

Of  the  real  nature  of  the  enzyme  nothing  is  known.  It  has  never 
been  isolated  in  a  condition  of  even  approximate  purity.  The  pan- 
creatic extracts  of  the  market  contain  the  enzymes  acting  on  fats  and 
carbohydrates  as  well,  in  addition  to  a  very  large  amount  of  other 
matter.  The  active  ferment  is  very  soluble  in  water  and  in  dilute 
glycerol  or  dilute  alcohol,  but  in  strong  alcohol  or  glycerol  it  is  insol- 
uble. In  presence  of  weak  hydrochloric  acid  trypsin  is  quickly  digested 
or  destroyed  by  pepsin,  and  at  temperatures  much  above  500  C.  it  soon 
becomes  inactive.  The  temperature  optimum  is  probably  about  40 ° 
to  450,  in  weak  alkaline  solution,  but  the  statements  in  the  literature 
on  this  point  are  somewhat  discrepant. 

Erepsin.  In  this  connection  another  peculiar  ferment,  which  in 
some  respects  resembles  trypsin,  must  be  mentioned.  Erepsin  is  found 
in  the  walls  of  the  small  intestine  and  is  concerned  in  the  final  splitting 


ENZYMES    AND    OTHER    FERMENTS.  I  I  I 

of  protein  derivatives.  It  acts  on  proteoses  and  peptones,  essentially, 
and  carries  the  hydrolysis  to  the  formation  of  comparatively  small 
amino  acid  groups,  that  is,  to  practically  complete  hydrolysis. 

THE  UREA   FERMENTATION. 
Urease  and  Other  Ferments.     Urine  exposed  to  the  air  soon 
becomes  alkaline  and  the  presence  of  ammonia  is  easily  shown.     This 
behavior  is  due  to  the  formation  of  ammonium  carbonate  from  the 
urea  by  a  reaction  which  may  be  expressed  in  this  way : 

(NH2)2CO  +  2H20  =  (NHJ2C03. 

The  agency  concerned  in  this  addition  of  water  molecules  was  for  a 
long  time  in  doubt,  but  investigation  finally  showed  it  to  be  a  case  of 
ferment  action.  In  all  urines  undergoing  this  change  numerous  bac- 
teria are  present  and  by  separating  and  making  pure  cultures  of  these, 
several  species  have  been  found  which  are  capable  of  decomposing  pure 
solutions  of  urea.  The  name  micrococcus  urecc  has  been  given  to  one 
of  the  most  active  of  these  bacterial  organisms.  It  has  been  found, 
however,  that  the  action  is  certainly  due  to  a  soluble  product  or  enzyme 
secreted  by  the  bacteria,  since  it  may  be  brought  into  solution.  This 
solution,  after  the  most  careful  filtration  even,  is  very  active  when 
properly  made  and  will  quickly  induce  the  ammoniacal  decomposition 
in  urea  solutions. 

The  name  urease  has  been  given  to  this  soluble  ferment,  which  must 
belong  to  the  hydrolytic  group.  It  is  active  up  to  about  500  C.  and  is 
much  more  stable  in  presence  of  alkalies  than  with  acids,  as  would  be 
supposed  from  the  reaction  it  produces.  The  enzyme  is  not  readily 
extracted  from  the  living  bacterial  cells ;  these  must  first  be  destroyed 
or  allowed  to  die  out  in  process  of  making  strong  cultures.  The  cells 
holding  this  enzyme  are  very  widely  distributed  in  nature,  being  found 
in  the  air,  in  most  soils  and  in  river  water.  This  accounts  for  the 
usually  rapid  fermentation  of  urine. 

B.     OXIDATION    REACTIONS. 

Under  the  head  of  oxidation  reactions  it  is  very  easy  to  include  some 
in  which  the  essential  phenomenon  is  clearly  one  of  addition  of  oxygen 
to  the  decomposing  substance.  This  is  certainly  the  case  in  the  pro- 
duction of  acetic  acid  from  weak  alcohol.  In  other  cases,  however,  the 
actual  nature  of  the  chemical  change  which  occurs  is  more  obscure  and 
the  classification  of  such  reactions  as  oxidation  reactions  is  possibly 
open  to  doubt,  as  will  appear  below.     In  some  cases  the  classification 


112  PHYSIOLOGICAL    CHEMISTRY. 

appears  very  arbitrary,  as  the  grouping  of  the  alcoholic  fermentation 
among  the  oxidation  processes  illustrates.  But  there  is  sufficient  rea- 
son for  this  to  justify  the  place  the  reaction  is  given. 

ALCOHOLIC    FERMENTATION. 

As  mentioned  at  the  outset  the  phenomena  of  alcoholic  fermentation 
were  the  first  to  claim  attention  and  many  of  the  fundamental  condi- 
tions were  empirically  established  long  before  the  part  played  by  yeast 
in  the  process  was  recognized.  After  the  investigations  of  Pasteur 
our  knowledge  in  this  field  rapidly  widened. 

Yeast.  The  common  agent  of  alcoholic  fermentation  is  known  as 
yeast,  but  under  this  term  are  included  a  very  large  number  of  really 
distinct  species.  In  fact  several  different  genera  may  be  and  actually 
are  employed  in  practice.  The  following  table  gives  an  idea  of  the 
relations  of  the  commoner  organisms  classed  among  the  alcoholic  fer- 
ments. The  yeasts  with  many  other  cells  are  classed  in  a  group  of  the 
budding  fungi,  or  Eumycetes,  as  distinguished  from  the  fission  fungi 
or  Schizomycetes. 

Family  Saccharomycetes 


Genus  Monospora        Saccharomyces        Schizosaccharomyces 

|"  Cerevisise 
Species  J  Ellipsoideus 

]  Pastorianus 
Land  others. 

A  few  molds,  also,  bring  about  alcoholic  fermentation.  We  have 
included  here  Mucor  mucedo,  M  tic  or  racemosus,  Mucor  Rouxii  and 
others  which  are  not  in  any  way  technically  useful. 

Ordinarily,  however,  we  take  as  the  type  of  a  yeast  the  common  beer 
yeast  Saccharomyces  cerevisice,  which  is  a  cultivated  species  employed 
in  fermentation  by  brewers  and  distillers.  In  the  natural  wine  fer- 
mentation other  species  seem  to  be  the  most  active.  These  are  found 
on  the  skin  of  the  grape  and  hence  find  their  way  into  the  juice  after 
crushing.  .S.  apiculatus  and  vS\  ellipsoideus  are  the  names  of  two  of 
the  most  important  of  the  species  active  in  this  way.  The  common 
beer  yeast  appears  in  the  form  of  nearly  spherical  cells  having  a  diam- 
eter of  8  to  9  /a.  It  is  active  through  a  comparatively  wide  range  of 
temperature.  In  practice  the  fermentation  of  malt  wort  to  produce 
beer  is  carried  out  at  a  very  low  temperature,  while  a  grain  mash  to 
produce  common  alcohol  or  whisky  is  fermented  at  a  high  temperature. 


ENZYMES    AND    OTHER    FERMENTS.  I  I  3 

In  the  one  case,  however,  weeks  are  required  to  complete  the  change, 
in  the  other  one  or  two  days. 

Like  most  similar  reactions  brought  about  by  living  cells  a  limit  to 
the  quantity  of  product  formed  is  soon  reached.  With  ordinary  glu- 
cose the  reaction  follows  approximately  according  to  this  equation : 

CeH^Oe  =  2C2H60  +  2C02. 

The  mechanism  of  the  reaction  is  not  known,  but  it  may  be  considered 
as  one  of  internal  oxidation,  since  the  carbon  of  the  liberated  gas  is  in 
the  fully  oxidized  condition. 

As  the  alcohol  formed  accumulates  a  point  is  reached  where  the 
activity  of  the  yeast  cell  is  impeded  and  the  fermentation  finally  stopped. 
This  occurs  when  about  20  per  cent  of  alcohol  has  accumulated  as  a 
reaction  product.  Very  strong  sugar  solutions  do  not  ferment  at  all. 
Indeed  some  of  the  common  uses  of  cane  sugar  in  preserving  fruits 
depend  on  this  fact.  Some  of  the  conditions  of  fermentation  may  be 
readily  illustrated  by  simple  experiments. 

Experiment.  Make  a  strong  cane  sugar  syrup,  by  boiling  or  heating  together  10 
gm.  of  sugar  and  10  cc.  of  water.  Allow  to  cool  and  add  about  a  gram  of  crumbled 
compressed  yeast,  and  then  set  aside  for  several  days.  The  solution  should  be 
found  free  from  any  signs  of  fermentation. 

Experiment.  Prepare  a  20  per  cent  solution  of  commercial  glucose  and  pour  50 
cc.  of  it  into  a  small  flask.  Add  some  yeast  and  allow  to  stand  two  days  in  a 
moderately  warm  place.  At  the  end  of  this  time  it  should  be  found  in  active 
fermentation,  as  shown  by  the  escape  of  gas  bubbles  and  the  odor  of  alcohol.  If 
allowed  to  stand  several  days  longer  in  the  ordinary  atmosphere  the  liquid  in  the 
flask  usually  becomes  sour  from  acetic  fermentation. 

Experiment.  Prepare  a  tube  with  sugar  solution  and  yeast  as  in  the  last  experi- 
ment. Close  it  loosely  with  a  plug  of  absorbent  cotton  and  heat  to  boiling,  allowing 
steam  to  escape  through  the  cotton.  If  the  tube  is  now  left  to  itself  for  several 
days  it  will  be  found  that  fermentation  has  not  taken  place,  showing  that  heat 
destroys  the  characteristic  property  of  the  yeast  cell. 

Experiment.  Prepare  another  tube  with  sugar  and  yeast  and  add  10  cc.  of  strong 
alcohol.  Shake  the  mixture  and  allow  to  stand.  No  fermentation  appears,  as  the 
activity  of  the  yeast  cell  is  destroyed  by  alcohol.  We  have  good  familiar  illustra- 
tions of  this  in  the  self-preservation  of  certain  "  heavy  "  wines,  as  ports,  sherries  and 
malagas,  while  "  light "  wines,  which  contain  10  to  12  per  cent  of  alcohol  usually, 
must  be  kept  tightly  bottled  for  preservation. 

Experiment.  Test  for  Alcohol.  We  have  many  tests  by  which  the  presence  or 
formation  of  alcohol  may  be  shown.  The  fermentation  of  a  saccharine  liquid  is 
followed  by  a  lowering  of  the  specific  gravity  as  may  be  easily  found  by  experiment, 
and  a  practical  quantitative  test  is  based  on  this  fact.  A  simple  chemical  test  for 
the  presence  of  alcohol,  which  in  most  cases  is  sufficient,  is  the  following:  Add  to  the 
clear  liquid  to  be  examined  a  few  small  crystals  of  iodine,  warm  to  about  60°  C, 
and  then  add  enough  sodium  hydroxide  or  sodium  carbonate  to  produce  a  colorless 
solution.  An  excess  of  the  alkali  must  not  be  used.  In  a  short  time  bright  yellow 
crystals  of  iodoform  precipitate,  easily  recognized  by  their  color  and  odor.  Certain 
other  liquids  give  the  same  test. 

9 


114  PHYSIOLOGICAL    CHEMISTRY. 

Ordinary  yeast  contains  the  soluble  ferment  called  invertase,  which 
has  been  already  referred  to.  This  may  be  shown  by  experiment, 
as  follows : 

Experiment.  Crush  some  yeast,  add  water  and  wash  by  decantation  or  on  a 
filter  thoroughly.  Then  rub  up  the  washed  yeast  with  some  water  in  a  mortar  and 
add  the  mixture  to  a  solution  of  pure  cane  sugar  which  has  previously  been  treated 
with  a  few  drops  of  a  strong  alcoholic  solution  of  thymol.  50  cc.  of  a  5  per  cent 
sugar  solution  will  answer.  The  thymol  prevents  the  action  of  the  yeast  cell  fermen- 
tation, but  does  not  prevent  the  action  of  the  invertase.  The  mixture  should  be 
kept  about  24  hours  at  a  temperature  of  400  to  500  C.  At  the  end  of  this  time  it 
is  filtered  and  the  filtrate  tested  for  invert  sugar  by  means  of  the  Fehling  solution. 
Ether  and  chloroform  are  sometimes  employed  in  place  of  the  thymol;  the  latter 
must  be  removed  by  heating  before  making  the  Fehling  test. 

Zymase.  It  has  been  intimated  already  that  the  activity  of  yeast 
as  an  alcoholic  ferment  is  due  to  the  presence  of  an  enzyme.  This 
fact,  long  suspected  and  much  debated,  was  finally  demonstrated  by  E. 
Buchner,  as  explained  above.  Buchner  rubbed  the  yeast  with  fine, 
sharp  sand  and  water  and  then  subjected  the  mixture  to  great  pressure. 
The  liquid  pressed  out  was  carefully  filtered  and  was  found  to  be  as 
active  as  the  original  yeast.  The  enzyme  in  it  he  called  zymase.  It 
clings  tenaciously  to  the  yeast  cell,  hence  the  necessity  of  destroying 
the  structure  by  grinding  with  sand,  and  employing  great  pressure. 

Zymase  is  not  a  very  stable  ferment  and  in  the  solution  obtained  is 
soon  destroyed  by  other  ferments  present.  The  yeast  extract  may, 
however,  be  concentrated  at  a  low  temperature  and  obtained  in  dry 
form  which  is  more  stable.  Extracts  made  from  yeast  by  simple  treat- 
ment with  water  may  contain  invertase  but  no  zymase.  It  seems  prob- 
able that  the  ferment  is  not  confined  to  the  yeast  cell.  It  has  long  been 
known  that  many  overripe  fruits  produce  a  small  amount  of  alcohol, 
even  when  the  possibility  of  the  presence  of  yeast  cells  is  entirely 
absent.  This  formation  of  alcohol  was  finally  ascribed  to  the  cell 
activity  of  the  fruits  themselves,  but  since  the  work  of  Buchner  it 
seems  more  rational  to  refer  the  appearance  of  alcohol  to  the  presence 
of  an  enzyme  in  the  ripe  fruit. 

It  should  also  be  said  that  sugar  may  be  made  to  yield  alcohol  by  a 
much  simpler  process.  It  has  been  found  that  a  sugar  solution  mixed 
with  a  little  potassium  hydroxide  and  placed  in  bright  sunlight  yields 
some  alcohol  and  carbon  dioxide.  This  is  of  course  a  purely  chemical 
decomposition,  and  suggests  the  possibility  of  chemical  reactions  in 
other  cases.  The  old  notion  as  to  the  necessity  of  the  presence  of 
living  cells  to  break  down  the  sugar  is  thus  completely  disproved. 


ENZYMES    AND    OTHER    FERMENTS.  I  I  5 

ACETIC   FERMENTATION. 

In  this  a  true  oxidation  takes  place,  the  oxygen  of  the  air  being 
employed  to  convert  weak  alcohol  into  the  acid  according  to  this 
reaction : 

CHcO  +  02  =  C2H402  +  H20. 

The  active  agent  concerned  in  the  fermentation  oxidation  is  the  cell 
found  in  "mother  of  vinegar." 

Mother  of  Vinegar  is  an  old  name  given  to  the  slimy  scum  or  sedi- 
ment which  forms  in  weak  alcoholic  liquids  that  turn  sour,  in  wine  or 
cider,  for  example.  Microscopic  examination  shows  this  substance  to 
consist  of  minute  cells  which  have  received  the  name  of  Micoderma 
a-ceti;  more  recently  the  name  Bacterium  accti  has  been  given  to  the 
plant  organism.  Thus  far  it  has  not  been  found  possible  to  isolate 
a  soluble  enzyme  from  the  cell  ferment.  One  may  be  present,  but 
attempts  to  obtain  it  have  failed. 

Besides  this  Bacterium  aceti  several  other  vinegar  ferments  are 
known.  Most  of  them  float  in  the  air,  and  when  lodged  in  a  weak 
alcohol  containing  certain  mineral  substances  produce  a  fermentation 
quickly.  A  dilute  aqueous  solution  of  pure  alcohol  will  not  ferment 
in  the  same  way;  the  presence  of  various  salts  and  organic  matters  in 
addition  is  necessary.  An  experiment  may  be  made  to  illustrate  vin- 
egar or  acetic  acid  fermentation. 

Experiment.  If  available  a  fruit  juice,  freshly  expressed  and  left  in  contact  with 
the  skin,  should  be  allowed  to  undergo  alcoholic  fermentation.  Or,  a  sugar  solution, 
as  described  some  pages  back,  may  be  allowed  to  ferment.  The  weak  alcoholic 
liquid  obtained  in  the  case  of  the  fruit  juice  will  next  turn  sour  from  the  pro- 
duction of  acetic  acid  by  the  action  of  the  germs  on  the  skin.  In  the  case  of 
the  alcohol  from  the  sugar  it  may  be  necessary  to  add  a  little  "  mother  of  vinegar  " 
from  a  vinegar  factory  to  induce  the  fermentation.  Presence  of  the  air  is  neces- 
sary to  complete  the  change.  The  acid  strength  of  the  product  may  be  finally  tested 
by  means  of  a  standard  alkali  solution  and  phenol-phthalein. 

THE  OXIDASE  ENZYMES. 
We  come  now  to  a  very  brief  consideration  of  an  obscure  but  inter- 
esting subject  about  which  our  knowledge  is  of  comparatively  recent 
origin.  In  certain  vegetable  substances  reactions  occur  which  are 
ascribed  to  the  presence  of  a  class  of  oxidizing  enzymes  called  oxidases. 
These  changes  are  illustrated  by  the  blackening  of  an  apple,  potato  or 
beet  which  is  cut  and  exposed  to  the  air.  The  cut  surfaces  soon  turn 
dark.  If  the  same  substances  are  thoroughly  heated  before  the  cutting 
the  color  change  does  not  follow.  Potato  or  apple  pulp  speedily 
darkens  in  the  air,  but  if  previously  cooked  the  natural  light  color  per- 


Il6  PHYSIOLOGICAL    CHEMISTRY. 

sists.  To  account  for  these  and  many  similar  reactions  it  has  been 
assumed  by  many  chemists  that  the  fruits  or  vegetables  in  question 
contain  an  oxygen-carrying  enzyme  and  at  the  same  time  some  chem- 
ical substance  on  which  this  can  act  with  the  production  of  color,  the 
oxygen  necessary  for  the  change  being  taken  from  the  air.  The 
action  of  this  enzyme  or  oxidase  may  be  shown  in  other  ways,  espe- 
cially by  the  use  of  hydroquinol  and  pyrogallol,  which  substances  yield 
very  dark  solutions  when  oxidized.  It  is  simply  necessary  to  make 
an  aqueous  extract  of  certain  vegetables  and  fruits  and  add  this  to 
the  aqueous  solution  of  the  hydroquinol  to  produce  the  dark  color. 
Here  the  enzyme  appears  to  be  active  enough  to  carry  oxygen  to  the 
hydroquinol. 

Laccase  and  Tyrosinase.  These  are  the  names  which  have  been 
given  to  two  of  these  oxidases.  The  first  was  originally  found  in  the 
sap  of  the  Japanese  lac  tree,  which  when  expressed  and  exposed  to  the 
air  darkens  and  produces  the  well-known  lacquer.  The  same  laccase 
is  said  to  be  one  of  the  agents  which  brings  about  the  darkening  in 
many  other  saps  and  juices.  Tyrosinase  acts  on  the  phenol  derivative 
tyrosine  which  is  found  in  traces  in  many  vegetables  and  causes  its 
oxidation. 

These  two  reactions  may  be  taken  to  represent  a  wide  range  of 
changes  in  which  phenol  bodies  are  concerned.  In  another  group  of 
reactions  aldehyde  bodies  are  turned  into  acids,  as  happens  to  salicylal- 
dehyde.  It  is  possible  that  many  of  the  obscure  oxidative  changes  of 
the  animal  body  are  brought  about  by  enzymes  of  this  type,  but  our 
knowledge  here  is  not  very  definite.  It  is  known  that  extracts  from 
the  liver  and  spleen  have  the  power  of  changing  hypoxanthine  and 
xanthine  to  uric  acid,  but  of  the  more  profound  oxidations  of  the  body 
much  less  is  known.  Cohnheim  has  described  a  glycolytic  ferment, 
active  in  the  combustion  of  sugar,  when  aided  by  a  co-enzyme,  or 
activator,  but  the  nature  of  the  change  is  not  one  which  can  be  clearly 
explained. 

Peroxidases.  Recently  the  term  peroxidase  has  been  introduced  to  describe  a 
peculiar  enzymic  ferment  which  occurs  in  animal  and  vegetable  cells,  the  striking 
feature  of  which  is  to  induce  the  oxidation  of  a  great  variety  of  substances  through 
hydrogen  peroxide.  Milk,  for  example,  when  fresh  has  the  power  of  bringing 
about  the  oxidation  of  phenol-phthalin  to  phenol-phthalein  by  hydrogen  peroxide, 
and  the  same  behavior  has  been  observed  in  other  animal  secretions.  The  intensity 
of  the  oxidation  is  much  increased  by  the  presence  of  various  other  substances 
which  serve  as  accelerators.  As  hydrogen  peroxide  is  very  readily  formed  by  a 
wide  range  of  reactions,  it  is  possible  that  it  is  produced  in  living  cells,  to  undergo 
immediate  destruction  through  the  activity  of  the  ferment  bodies.  This  may  have 
some  bearing  on  the  explanation  of  animal  oxidations,  but  as  yet  our  knowledge 
on  this  point  is  scarcely  beyond  the  speculative  stage. 


ENZYMES    AND    OTHER    FERMENTS.  117 

C.     BACTERIOLYTIC    PROCESSES. 

The  term  bacteriolytic  is  applied  to  such  fermentation-splitting  proc- 
esses as  may  be  carried  out  by  bacteria  without  the  addition  of  oxygen. 
In  the  acetic  acid  fermentation,  which  is  likewise  a  bacterial  process, 
the  presence  of  oxygen  is  necessary,  but  there  are  several  somewhat 
analogous  reactions  in  which  oxygen  is  not  required  and  these  are 
included  in  the  present  group.  It  must  be  admitted  of  course  that  the 
division  is  a  perfectly  artificial  one  based  on  convenience  rather  than 
on  marked  differences  in  agents  or  products.  Some  of  the  reactions 
classed  here  have  long  been  described  as  fermentations  and  have  been 
studied  in  connection  with  the  other  common  ferment  changes.  These 
will  be  taken  up  first.  But  we  have,  in  addition,  further  changes  which 
are  certainly  of  the  same  general  character  and  call  for  like  treatment. 

LACTIC  AND  BUTYRIC  FERMENTATIONS. 

Why  milk  turns  sour  spontaneously  in  warm  weather  is  an  old 
question,  but  it  was  not  satisfactorily  answered  until  after  the  time  of 
Pasteur's  pioneer  labors.  Following  his  work  on  the  yeasts  Pasteur 
took  up  other  problems  of  fermentation  and  pointed  out  the  general 
nature  of  the  reaction  by  which  the  sour  substance  present,  lactic  acid, 
is  formed.  He  found  the  production  of  lactic  acid  to  depend  on  the 
ferment  activity  of  certain  microorganisms,  which  have  later  been 
more  fully  described  by  bacteriologists. 

Lactic  Acid  Bacteria.  It  was  found  that  lactic  acid  is  formed 
from  the  simple  sugars  by  a  splitting  process  which  for  a  long  time 
was  illustrated  by  an  equation  supposed  to  represent  the  facts  quanti- 
tatively : 

C6H1=O0  =  2C3H0O3. 

It  was  also  recognized  that  not  merely  one,  but  many  species  of  bac- 
teria are  capable  of  decomposing  sugar  solutions  in  this  way.  Of  these 
the  form  known  as  Bacillus  acidi  lactici  has  been  perhaps  the  most 
thoroughly  studied ;  it  appears  to  be  always  present  in  milk  which  has 
soured  spontaneously,  and  can  be  found  in  the  air,  especially  of  pas- 
tures or  cowsheds.  Many  soils  also  contain  the  organism.  In  no 
case,  however,  is  the  reaction  a  perfectly  sharp  one;  along  with  the 
lactic  acid  other  products  are  formed,  acetic  acid,  alcohol,  formic  acid, 
carbon  dioxide  and  hydrogen  being  the  most  common.  In  some  cases 
the  proportion  of  lactic  acid  is  relatively  small.  The  formation  of 
lactic  acid  may  be  illustrated  by  a  laboratory  experiment. 

Experiment.  To  ioo  cc.  of  20  per  cent  cane  sugar  solution  add  an  equal  volume 
i/f  aqueous  malt  extract  and  10  to  15  grams  of  precipitated  chalk.     Inoculate  this 


Il8  PHYSIOLOGICAL    CHEMISTRY. 

mixture  with  a  culture  of  lactic  acid  bacteria  and  keep  at  a  temperature  of  about 
400  C.  for  some  days.  The  chalk  is  necessary  to  take  up  the  acid  as  fast  as 
formed;  without  it  the  fermentation  soon  ceases,  as  the  ferment  is  extremely  sensi- 
tive to  the  action  of  free  acid.  The  mixture  must  be  shaken  from  time  to  time. 
As  the  fermentation  progresses  the  slightly  soluble  calcium  lactate  begins  to  sepa- 
rate. In  a  good  fermentation  enough  of  this  forms  to  fill  the  fermenting  vessel 
with  a  mass  of  crystals.  These  crystals  are  redissolved  in  hot  water,  and  the  solu- 
tion filtered.  The  filtrate  on  concentration  deposits  crystals  of  calcium  lactate, 
Ca(C3H503)2.sH20,  which  may  be  collected  and  dried  between  folds  of  filter  paper. 
The  free  lactic  acid  may  be  obtained  by  decomposing  the  calcium  salt  with  sul- 
phuric acid  in  the  proper  amount  and  shaking  out  with  repeated  small  portions  of 
ether.  The  lactic  acid  dissolves  in  the  ether  and  is  left  when  this  is  evaporated. 
Zinc  oxide  may  be  employed  in  place  of  calcium  carbonate  to  neutralize  during 
the  fermentation.  In  this  case  zinc  lactate  forms,  from  which  the  acid  may  be 
separated  by  dissolving  the  crystals  in  hot  water  and  decomposing  the  solution  by 
means  of  hydrogen  sulphide. 

Several  pure  cultures  of  lactic  acid  bacteria  can  now  be  obtained  for  technical  use. 
For  the  rapid  production  of  the  acid  Lafar  recommends  Bacillus  acidificans 
longissimus. 

Pure  lactic  acid  as  prepared  by  fermentation  is  a  thickish  liquid,  with 
marked  acid  taste  and  but  slight  odor.  It  is  optically  inactive,  but  may 
be  resolved  into  active  components  by  treatment  with  strychnine,  which 
crystallizes  with  the  levo  modification.  This  common  fermentation 
acid  is  employed  for  several  purposes  in  the  industries  and  is  now  com- 
paratively cheap  since  the  introduction  of  methods  of  fermentation 
with  pure  cultures. 

Lactic  acid  fermentations  are  concerned  in  many  common  opera- 
tions. In  the  leavening  of  bread  along  with  yeast  fermentation  there 
is  usually  a  bacterial  fermentation  with  production  of  acid.  In  some 
kinds  of  bread  this  is  extremely  important.  In  the  preparation  of 
sauerkraut  and  many  pickles  a  lactic  acid  fermentation  is  the  charac- 
teristic feature.  Several  well-known  beverages  produced  from  milk 
are  fermented  in  such  a  manner  that  they  contain  lactic  acid;  kephir 
and  kumyss  are  illustrations.  Yeasts  and  the  lactic  acid  bacteria  work 
together  in  many  instances  and  symbiotic  products  are  the  rule,  per- 
haps, rather  than  the  exception  in  fermentations.  In  the  milk  indus- 
tries these  mixed  fermentations  are  apparently  essential  in  the  ripen- 
ing processes,  and  in  certain  distillery  fermentations  with  yeast  a. lactic 
acid  fermentation  is  encouraged  to  prevent  the  growth  and  action  of 
the  bacteria.  This  fermentation  lactic  acid  is  found  also  in  the  stomach 
and  the  intestine.  In  the  stomach  the  formation  of  any  large  amount 
is  usually  impossible  because  of  the  presence  of  hydrochloric  acid. 
About  o.i  per  cent  of  free  hydrochloric  acid  is  sufficient  to  impede  the 
lactic  fermentation.  Free  mineral  acids  are  not  present  in  the  intes- 
tine; the  organic  fermentation  acids  may  therefore  be  formed  in  appre- 


ENZYMES    AND    OTHER    FERMENTS.  119 

ciable  quantities.  Fermentation  lactic  acid  must  not  be  confounded 
with  the  isomeric  sarcolactic  acid  found  in  the  muscles. 

Butyric  Acid  Fermentations.  Another  very  important  kind  of 
acid  fermentation  is  that  which  results  in  the  formation  of  normal 
butyric  acid : 

CeH3208  =  2H2  +  2C02  -f  C4H802. 

As  in  the  case  of  lactic  acid  this  butyric  acid  fermentation  is  not  the 
result  of  the  action  of  one  organism  only,  but  it  may  be  produced  by 
several,  and  furthermore  several  by-products  are  always  produced  in 
quantity.  The  above  reaction  is  then  merely  a  limit  reaction,  which  is 
approached  but  never  absolutely  realized. 

In  the  milk  fermentation  the  lactic  acid  or  calcium  lactate  formed 
may  be  further  changed  to  butyric  acid,  the  necessary  ferment  entering 
from  the  air.  Most  river  waters  contain  butyric  acid  bacteria,  which 
bring  about  the  characteristic  fermentations  when  the  water  is  mixed 
with  some  sterilized  milk,  as  in  one  of  the  common  tests  carried  out  in 
the  sanitary  examination  of  water.  Garden  soils  are  also  rich  in  some 
of  these  butyric  acid-producing  organisms,  and  may  be  used  in  starting 
a  fermentation,  as  may  be  illustrated  in  this  way: 

Experiment.  Mix  100  cc.  of  a  5  per  cent  glucose  solution  with  four  or  five 
grams  of  fibrin  and  heat  to  boiling.  To  the  hot  solution  add  a  few  grams  of  garden 
loam  and  allow  the  liquid  to  cool  rapidly.  The  bacterial  spores  resist  the  heat 
while  other  forms  succumb  and  are  thus  disposed  of.  Keep  the  mixture  at  a  tem- 
perature of  about  27°  to  400  C.  Fermentation  begins  in  about  two  days  and  is 
assisted  by  neutralizing  with  a  little  sodium  hydroxide  from  time  to  time.  After 
several  days  the  presence  of  butyric  acid  may  be  shown  by  warming  some  of  the 
liquid  with  sulphuric  acid,  or  with  sulphuric  acid  and  alcohol.  In  the  latter  case 
the  odor  of  ethyl  butyrate  formed  is  very  characteristic. 

Butyric  acid  in  pure  condition  is  a  strongly  acid  liquid  possessing  a 
rather  disagreeable  odor.  It  is  frequently  present  in  the  stomach,  but 
its  occurrence  there  is  really  abnormal.  If  the  gastric  juice  contains 
the  proper  amount  of  hydrochloric  acid  a  butyric  acid  fermentation  is 
not  possible.  With  diminished  hydrochloric  acid,  however,  bacterial 
fermentations  can  take  place.  In  the  arts,  while  lactic  fermentation 
is  desirable  frequently,  and  encouraged,  the  butyric  fermentation  is 
usually  considered  very  objectionable  and  is  prevented  if  possible. 

Other  Fermentations.  It  will  not  be  necessary  to  explain  at 
length  any  other  cases  of  bacterial  fermentations,  as  these  two  just 
given  are  sufficient  for  illustration.  What  is  known  as  the  mucous 
fermentation  sometimes  takes  place  in  saccharine  liquids  or  in  wines 
which  have  not  been  completely  fermented.  A  slimy  mucilaginous 
product  is  formed  here  which  contains  a  kind  of  gum.     Certain  micro- 


120  PHYSIOLOGICAL    CHEMISTRY. 

organisms  have  the  power  of  decomposing  cellulose  and  the  operation 
is  called  the  cellulose  fermentation.  The  products  of  this  reaction 
with  certain  bacteria  are  mainly  gaseous,  hydrogen  and  marsh  gas  pre- 
dominating. Certain  organisms  are  able  to  produce  fatty  acids  also. 
In  the  intestines  of  the  herbivora  changes  of  this  character  take  place, 
and  the  acids  produced  are  doubtless  of  value  in  aiding  in  some  of  the 
other  digestive  processes  which  take  place  there. 


CHAPTER   VII. 

SALIVA   AND    SALIVARY    DIGESTION. 

It  has  already  been  said  that  the  saliva  contains  an  enzyme  known 
as  ptyalin,  the  function  of  which  is  to  begin  the  digestion  of  starchy 
foods.  It  remains  now  to  look  into  the  nature  of  this  process  a  little 
more  closely,  and  to  study  the  conditions  of  this  kind  of  fermentation. 
The  saliva  as  secreted  by  the  three  large  pairs  of  glands  of  the  mouth 
is  a  thin  liquid  with  slightly  alkaline  reaction.  Because  of  the  constant 
presence  of  mucus  and  epithelial  cells  it  is  never  clear  but  presents 
always  an  opalescent  appearance.  The  amount  secreted  daily  varies 
between  i  and  2  liters.  In  the  last  few  years  Pawlow  has  shown  how 
a  normal  saliva  may  be  collected  from  animals. 

In  the  older  literature  several  complete  analyses  of  saliva  are  given, 
but  less  importance  is  now  attached  to  these  than  formerly,  since  a 
great  degree  of  exactness  is  not  possible  in  such  tests  and  besides  the 
composition  of  the  secretion  cannot  be  a  constant  one.  In  the  mean 
the  amount  of  water  present  is  99.5  per  cent.  In  the  0.5  per  cent  of 
solids  about  0.2  per  cent  consists  of  inorganic  salts  and  the  remainder 
of  organic  substances,  including  the  ferment.  Among  the  salts  there 
is  a  minute  trace  of  potassium  thiocyanate,  KSCN,  which  may  fre- 
quently be  recognized  by  the  test  with  ferric  chloride.  It  is  not  known 
that  this  substance  exerts  any  specific  function,  and  in  different  indi- 
viduals it  is  present  in  different  amounts.  Some  of  the  important 
properties  of  saliva  may  be  illustrated  by  simple  experiments. 

Experiment.  After  washing  out  the  mouth  thoroughly  with  water  chew  a  piece 
of  rubber  or  other  neutral  insoluble  substance  to  stimulate  the  flow  of  saliva. 
Collect  25  to  50  cc.  in  a  clean  beaker  and  after  diluting  with  an  equal  volume  of 
distilled  water  allow  to  stand  a  short  time  to  settle.  Then  filter  through  a  small 
filter  paper  into  a  clean  vessel  and  use  the  filtrate  for  the  following  tests : 

To  a  few  cc.  of  the  clear  saliva  in  a  test-tube  add  several  drops  of  a  dilute  solu- 
tion of  ferric  chloride.  This  gives  a  more  or  less  marked  red  color  from  the 
formation  of  ferric  thiocyanate.  A  very  strong  reaction  must  not  be  expected. 
Make  a  comparative  test  by  adding  a  like  amount  of  ferric  chloride  to  dilute 
solutions  of  potassium  thiocyanate. 

The  addition  of  solution  of  mercuric  chloride  discharges  the  color.  This  test 
is  of  value  in  distinguishing  between  a  thiocyanate  and  a  meconate,  which  sometimes 
has  value  in  medico-legal  work. 

Test  the  reaction  of  saliva  with  neutral  litmus  paper.  It  will  be  found  slightly 
alkaline.     Now  add  two  or  thre<    drops  of  dilute  acetic  acid  and  note  that  a  stringy 

121 


122  PHYSIOLOGICAL    CHEMISTRY. 

precipitate  of  mucin  separates.  Filter  off  this  precipitate  and  test  the  filtrate  for 
proteins  by  boiling  with  Millon's  reagent  or  by  the  xanthoproteic  reaction. 

Make  a  thin  starch  paste,  about  a  gram  to  200  cc.  of  water,  and  observe  that  it 
does  not  respond  to  the  Fehling  sugar  test  already  described.  Mix  10  cc.  of  this 
paste  with  5  cc.  of  the  filtered  saliva  and  warm  to  a  temperature  not  above  400  C. 
for  about  15  minutes.  At  the  end  of  this  time  apply  the  sugar  test  again.  A 
yellow  or  red  precipitate  will  appear  now,  showing  that  the  starch  has  been  con- 
verted, in  part  at  least,  into  sugar. 

The  saliva  alone  fails  to  reduce  the  copper  solution,  as  should  be  shown  by  trial. 

Pour  about  5  cc.  of  the  clear  saliva  into  a  test-tube  and  boil  a  few  minutes; 
add  the  starch  paste  and  allow  to  stand  as  in  the  above  experiment.  On  testing 
with  the  copper  solution  no  sugar  will  be  found,  showing  that  heat  destroys  the 
activity  of  the   ferment. 

The  digesting  power  of  the  saliva  is  destroyed  also  by  the  addition  of  a  small 
amount  of  strong  acid  or  alkali  solution,  which  the  student  should  prove  by 
experiment. 

Saliva  is  practically  without  action  on  raw  starch,  as  may  be  shown  in  this 
way.  Stir  a  small  amount  of  uncooked  potato  starch  into  5  cc.  of  saliva,  and  allow 
to  stand  15  minutes  at  3S°-40°,  and  filter.  Now  apply  the  Fehling  test,  and  note 
the  absence  of  precipitated  copper  suboxide. 

THE  CONVERSION  OF  STARCH. 

The  action  of  ptyalin  on  starch  is  a  complicated  one  and  in  all  details 
cannot  be  satisfactorily  described.  In  many  respects  the  digestive 
behavior  of  the  enzymes  of  the  saliva  and  of  malt  is  similar  to  that  of 
weak  acid.  The  complex  insoluble  starch  molecule  is  in  some  manner 
broken  up  and  partly  soluble  bodies  result.  This  change  is  at  first 
unaccompanied  by  hydration,  but  later  the  normal  enzymic  reaction  of 
water  addition  follows  and  the  dextrin  bodies  first  produced  become 
sugars.  Malt  sugar  is  formed  first,  and  in  the  case  of  acids  this  gives 
rise  finally  to  glucose  by  further  conversion.  But  with  ptyalin  the 
main  action  seems  to  end  with  the  production  of  maltose;  at  all  events 
no  large  amount  of  the  hexose  sugar  is  formed.  A  little  maltase  is 
said  to  be  present.  Furthermore  the  whole  of  the  starch  is  not  brought 
into  the  sugar  condition;  a  portion  remains  in  the  form  of  a  dextrin. 
In  an  earlier  chapter  something  was  said  about  the  character  of  these 
dextrins. 

In  most  respects  the  behavior  of  ptyalin  is  very  similar  to  that  of 
malt  diastase,  which  can  be  shown  by  a  simple  experiment  with  com- 
mercial malt.  This  substance  is  usually  made  by  germinating  barley 
and  permitting  the  growth  to  continue  some  days,  the  barley  in  moist 
condition  being  spread  out  on  a  so-called  malting  floor  to  encourage 
the  growth  and  prevent  overheating.  In  the  germination  the  enzyme 
is  developed,  probably  from  a  portion  of  the  protein  substance  present. 
When  the  action  has  gone  far  enough,  which  the  malster  recognizes 


SALIVA    AND    SALIVARY    DIGESTION.  1 23 

by  the  appearance  of  the  rootlet  thrown  out,  the  action  is  checked  by 
quick  drying,  leaving  the  diastase  in  permanent  stable  condition.  This 
malt  is  made  in  enormous  quantities  for  use  in  breweries  and  distil- 
leries. In  the  germinating  seed  in  the  ground  the  same  enzyme  is 
formed  which  converts  starch  into  soluble  food  for  the  young  plant. 

Experiment.  Mix  about  10  gm.  of  pale  ground  malt  with  50  cc.  of  lukewarm 
water,  and  allow  the  mixture  to  stand  a  short  time,  with  frequent  stirring  and 
shaking.  Then  filter  and  add  the  clear,  bright  filtrate  to  a  thin  starch  paste  made 
of  10  grams  of  starch  with  250  cc.  of  water.  The  starch  paste  must  be  cool  when 
the  malt  extract  is  added.  Place  the  mixture  on  the  water-bath  and  warm  to  500- 
6o°  C,  and  maintain  this  temperature.  Note  that  the  liquid  gradually  becomes 
thin  and  transparent.  From  time  to  time  remove  a  few  drops  by  means  of  a 
pipette,  and  test  with  iodine  solution.  At  first  a  deep  blue  color  appears,  but  this 
grows  weaker,  giving  place  to  violet,  then  to  yellowish  brown  and  finally  no  color 
is  obtained,  indicating  completion  of  the  reaction.  The  starch  paste  is  first  con- 
verted into  dextrin  and  finally  into  maltose.  Evaporate  the  solution  to  a  very 
small  volume  and  observe  the  taste  and  appearance  of  the  residue.  In  the  end 
product  there  is  usually  about  80  per  cent  of  maltose  and  20  per  cent  of  dextrin 
when  made  at  the  temperature  of  this  experiment. 

It  has  been  found  in  practice  that  the  amount  of  malt  sugar  formed 
depends  on  the  temperature  and  duration  of  the  digestion  with  diastase. 
At  a  lower  temperature  with  longer  action  the  conversion  of  the  dextrin 
becomes  more  perfect.  This  corresponds  with  the  behavior  of  the 
pancreatic  diastase  which  is  active  through  a  longer  period  usually  than 
is  possible  with  the  saliva. 

BEHAVIOR  OF  THE  DIASTASE. 

The  question  of  the  identity  of  the  malt  diastase  with  that  from 
saliva  is  still  a  disputed  one ;  while  some  writers  describe  them  as  iden- 
tical, others  apparently  find  characteristic  points  of  difference.  The 
behavior  of  saliva  with  various  reagents  has  been  pretty  thoroughly 
studied;  stronger  acids  and  alkalies  have,  of  course,  a  destructive 
action,  but  experiments  seem  to  show  that  very  weak  acids  favor  rather 
than  retard  the  digestive  power.  When  the  acid  strength  is  gradually 
increased  up  to  that  of  the  gastric  juice,  the  effect  of  the  ptyalin  on 
starch  paste  grows  weaker  and  finally  becomes  zero  long  before  the 
maximum  acidity  is  reached.  In  the  mouth  the  action  of  the  saliva  is 
certainly  largely  mechanical,  since  the  time  for  any  other  action  is 
entirely  too  short,  but  with  the  passage  of  the  food  into  the  stomach  it 
does  not  follow  that  all  diastatic  digestion  ceases  because  of  the  acid 
condition  of  that  organ.  After  the  beginning  of  a  meal  some  time  is 
required  for  the  commencement  of  hydrochloric  acid  secretion,  and  a 
further  time  before  enough  has  accumulated  to  seriously  interfere  with 
the  activity  of  the  diastase.     The  effect  of  the  acid  is  dependent  on  its 


124  PHYSIOLOGICAL    CHEMISTRY. 

concentration,  not  on  the  gross  amount  present.  Up  to  a  concentration 
of  about  o.oi  per  cent  the  acid  seems  to  have  but  little  inhibiting  action. 
Therefore  while  this  amount  of  free  acid  is  accumulating  we  may  sup- 
pose the  salivary  digestion  to  go  on  in  the  stomach.  Later,  with 
increase  in  acid,  the  ptyalin  disappears,  possibly  through  gastric 
digestion. 

Many  salts  exert  an  influence  on  the  rate  of  diastatic  digestion. 
Usually  this  is  to  retard  the  action,  but  sodium  chloride  and  other 
neutral  salts  in  small  amount  have  a  beneficial  effect.  With  other  sub- 
stances the  action  is  generally  unfavorable.  Small  amounts  of  protein 
matter,  or  preferably  the  syntonin  or  acid  albumins  formed  from  the 
proteins  by  combination  with  traces  of  hydrochloric  acid,  seem  to 
increase  slightly  the  activity  of  the  salivary  diastase.  This  is  a  point 
of  considerable  importance  in  explaining  possibly  the  continuation  of 
the  ptyalin  reaction  in  the  stomach.  Acid  combined  with  protein 
behaves  as  free  acid  toward  certain  indicators,  while  with  other  indi- 
cators it  does  not  show.  Starch  digestion  with  saliva  in  a  mixture 
containing  protein  and  hydrochloric  acid,  as  indicated  by  dimethyla- 
minoazobenzene,'  cannot  continue,  but  if  the  indication  is  merely  by 
phenol-phthalein  the  ptyalin  action  may  still  go  on,  since  in  this  case 
the  acid  shown  may  possibly  be  wholly  or  largely  combined  with  pro- 
tein substances.  Recent  investigations  have  shown  that  under  such 
conditions,  which  are  probably  duplicated  in  the  stomach,  the  digestion 
of  starch  may  go  on  at  practically  the  normal  rate,  the  hydrochloric 
acid  being  rapidly  combined  with  protein,  and  therefore  comparatively 
inert  with  ptyalin.  The  alkalinity  of  human  saliva  is  usually  referred 
to  as  due  to  the  presence  of  sodium  carbonate,  but  soluble  phosphates 
are  present  which  may  account  for  the  reaction  as  shown  by  certain 
indicators,  especially  by  litmus.  With  phenol-phthalein  the  reaction 
appears  neutral  ordinarily  or  even  slightly  acid.  With  the  latter  sub- 
stance as  indicator  it  is  generally  necessary  to  add  a  little  alkali  to 
secure  neutrality.  With  litmus  as  indicator  the  average  alkalinity, 
expressed  in  terms  of  Na2COs,  is  0.15  per  cent.  This  reaction  seems 
to  vary  with  the  time  of  day  and  is  strongest  before  breakfast.  Although 
carbon  dioxide  is  present  in  saliva,  it  probably  occurs  as  bicarbonate 
rather  than  as  carbonate,  which  would  account  for  the  reactions 
noticed. 

Many  soluble  substances  introduced  into  the  blood  in  any  way  soon 
appear  in  the  saliva.  This  may  be  shown  by  an  experiment  which 
illustrates  also  the  rapidity  of  absorption. 


SALIVA    AND    SALIVARY   DIGESTION.  1 25 

Experiment.  Swallow  about  a  gram  of  potassium  iodide  in  a  gelatin  capsule. 
In  this  manner  the  salt  is  gradually  dissolved  in  the  stomach  without  having  come 
in  direct  contact  with  the  mouth.  After  a  few  minutes  begin  testing  the  saliva  for 
iodine.  At  first  the  tests  are  all  negative,  but  in  time  a  reaction  appears  on  treat- 
ing the  saliva  with  something  to  liberate  the  iodine  in  presence  of  starch  paste. 
Solution  of  sodium  hypochlorite  may  be  used  for  this  purpose.  The  time  required 
to  exhibit  this  absorption  and  secretion  with  the  saliva  varies  greatly  in  different 
individuals. 


CHAPTER   VIII. 

THE   GASTRIC  JUICE  AND   CHANGES   IN   THE   STOMACH. 

The  gastric  juice  free  from  saliva  and  particles  of  food  is  a  thin 
liquid  with  specific  gravity  ranging  from  i.ooi  to  i.oio.  It  contains 
besides  certain  enzymes  some  small  amounts  of  protein  matters,  a  little 
sodium  chloride  and  traces  of  other  salts  and  free  hydrochloric  acid. 
Lactic  acid  is  also  frequently  present  in  traces.  The  older  analyses  of 
human  gastric  juice,  which  have  been  frequently  quoted,  are  mislead- 
ing, as  they  were  made  with  material  containing  saliva  and  food 
products.  By  aid  of  a  fistula  it  has  been  possible  to  obtain  a  fairly 
normal  secretion  from  certain  animals,  especially  from  the  dog,  and 
much  of  our  knowledge  of  the  conditions  of  secretion  and  variations 
in  composition  has  been  secured  in  this  way.  In  this  direction  the 
work  of  Pawlow  has  been  of  the  greatest  importance,  and  his  experi- 
ments have  given  us  new  ideas  on  the  subject  of  the  gastric  secretion. 
The  physiologically  important  substances  in  the  gastric  juice  are  free 
hydrochloric  acid,  pepsin,  rennin,  and  a  lipase. 

The  secretion  is  furnished  by  two  kinds  of  glands  known  as  the 
pyloric  glands  and  the  fundus  glands.  Both  groups  of  cells  yield  the 
two  enzymes,  but  the  pyloric  cells  do  not  seem  to  furnish  an  acid  secre- 
tion. It  is  probable  that  certain  of  the  fundus  cells  only  are  concerned 
with  the  acid  secretion.  The  gastric  secretion  is  promoted  by  two 
kinds  of  stimuli.  Certain  chemical  substances  when  taken  into  the 
stomach  have  the  power  of  exciting  a  flow  of  the  juice  from  the  mucous 
membrane,  and  are  themselves  not  subject  to  gastric  digestion.  Small 
amounts  of  alcohol,  ether,  spices  and  meat  extracts  act  in  this  way. 
But  more  important  than  this  is  the  so-called  "psychic"  stimulus, 
depending  on  the  desire  for  food  and  the  satisfaction  derived  from 
partaking  of  it.  The  amount  of  the  secretion  varies  with  the  nature 
and  kind  of  food. 

THE  DIGESTIVE  AGENTS. 

Origin  of  the  Free  Hydrochloric  Acid.  The  material  from  which 
the  fundus  cells  produce  the  enzymes  and  the  acid  is  the  blood.  But 
this  is  always  slightly  alkaline  and  to  account  for  the  secretion  of  a 
characteristic  acid  from  such  a  source  has  long  been  a  puzzle  to  physi- 
ologists.    Several  hypotheses  have  been  advanced,  but  these  are  all 

126 


THE  GASTRIC  JUICE  AND  CHANGES  IN   THE  STOMACH.  12J 

more  or  less  faulty.  The  difficulty  is  not  with  the  liberation  of  hydro- 
chloric acid,  which  is  a  purely  chemical  question,  and  one  which  may 
now  be  explained,  but  with  its  secretion. 

The  blood  contains  always  a  small  amount  of  sodium  chloride  and 
an  excess  of  carbonic  acid.  In  a  solution  containing  these  two  things 
some  double  decomposition  must  take  place  with  production  of  a  little 
free  hydrochloric  acid  and  sodium  carbonate.  According  to  the  older 
view  hydrochloric  acid  is  so  much  stronger  than  carbonic  acid  that  the 
liberation  of  the  former  from  a  chloride  by  the  action  of  the  latter  is 
impossible.  But  this  view  leaves  out  of  consideration  the  effect  of 
a  much  greater  mass  of  the  weaker  acid  through  which  in  fact  a  disso- 
ciation of  the  chloride  is  to  a  certain  extent  accomplished.  But, 
granting  this  kind  of  a  double  decomposition,  it  is  still  beyond  our 
powers  to  explain  how  the  free  acid  formed  in  the  cells  is  able  to  pass 
in  one  direction  into  the  stomach,  while  the  sodium  carbonate  produced 
at  the  same  time  wanders  in  the  other  direction  into  the  blood. 

This  acid  is  not  liberated  in  constant  amount  at  all  times  but  its  flow 
is  subject  to  the  influence  of  the  various  stimuli  referred  to  above. 
The  quantity  present  then  in  the  stomach  may  vary  from  a  mere  trace, 
or  zero  even,  to  a  maximum.  This  maximum  may  be  0.5  or  0.6  per 
cent  of  the  liquid  contents.  It  has  usually  been  given  as  much  lower. 
How  it  is  measured  will  be  shown  below.  Just  what  is  meant  by  the 
term  free  hydrochloric  acid  will  be  presently  explained. 

The  Enzymes.  In  an  earlier  chapter  the  general  nature  and  beha- 
vior of  the  three  gastric  ferments,  the  pepsin,  rennin  and  the  lipase  was 
pointed  out.  Whether  the  first  two  bodies  are  always  secreted  simul- 
taneously and  in  corresponding  amounts  is  not  definitely  known,  but 
that  this  is  the  case  is  often  assumed;  it  will  be  recalled  that  the  fol- 
lowers of  the  Pawlow  school  consider  the  enzymes  identical,  as  referred 
to  above.  In  fact,  one  of  the  clinical  methods  in  use  for  the  estimation 
of  "peptic"  activity  is  based  on  the  measurement  of  the  rennet  action 
through  milk  coagulation.  The  process  seems,  however,  of  doubtful 
value.  In  speaking  of  gastric  digestion  in  adults  we  are  concerned 
mainly  with  what  takes  place  through  the  action  of  pepsin,  which  will 
now  be  discussed.     A  briefer  discussion  of  the  other  ferments  will 

follow. 

PEPTIC    DIGESTION. 

In  presence  of  free  acids  of  the  so-called  "stronger"  type  pepsin 
has  the  power  of  effecting  remarkable  changes  in  protein  substances, 
which  have  been  the  subject  of  numerous  investigations.  In  the 
stomach  hydrochloric  acid  only  comes  into  play  and  it  first  gradually 


128  PHYSIOLOGICAL    CHEMISTRY. 

converts  the  proteins  present  into  acid-albumins  or  syntonin  bodies. 
This  is  the  preliminary  stage  in  the  digestion  of  these  food  substances 
and  must  be  accomplished  before  the  other  steps  in  the  stomach  are 
possible. 

In  this  reaction  the  hydrochloric  acid  enters  into  a  kind  of  combi- 
nation with  the  protein.  The  product  has  just  been  spoken  of  as  acid 
albumin,  but  it  is  evidently  through  the  basic  character  of  the  protein 
complex  that  the  combination  can  take  place.  The  protein  here  is  in 
effect  a  very  weak  base.  The  amount  of  acid  which  may  be  so  held 
is  considerable,  and  may  in  fact  amount  to  5  per  cent  or  more  of  the 
weight  of  the  protein.  With  certain  of  the  derived  protein  products 
the  weight  of  hydrochloric  acid  combined  is  even  larger,  at  times  as 
much  as  15  per  cent  of  the  protein  weight  being  so  held.  These 
derived  products  are  hydrolysis  products  with  smaller  molecular  weight 
and  evidently  more  available  amino  groups  to  hold  the  acid. 

It  is  generally  held,  as  just  stated,  that  this  acid  fixation  is  the  first 
step  in  the  gastric  digestion,  although  some  authors  claim  to  have  rec- 
ognized the  albumose  stage  as  the  primary  one  in  some  cases.  While 
this  acid  reaction  may  take  place  in  pure  aqueous-acid  solution,  it  is 
much  more  quickly  reached  in  presence  of  pepsin,  as  is  the  case  in  the 
stomach.  Experiments  with  artificial  mixtures  show  that  the  combi- 
nation then  is  almost  immediate,  as  is  made  evident  by  the  loss  of 
"  free  "  acidity,  to  be  explained  below.  Then  the  hydrolysis  goes  on 
and  the  various  derived  products  mentioned  in  a  former  chapter  are 
produced.  In  the  gastric  digestion  it  is  likely  that  the  cleavage  does 
not  usually  extend  beyond  the  production  of  the  secondary  albumoses ; 
that  is,  not  much  real  peptone  is  formed  in  the  time  naturally  con- 
sumed in  normal  digestion.  In  practice  the  larger  part  of  the  peptone 
production  is  doubtless  left  for  the  trypsin  conversion. 

Hydrolytic  cleavage  beyond  the  acid  albumin  stage  is  favored  by 
abundance  of  free  acid,  but  in  absence  of  this  it  still  goes  on.  In 
actual  digestion  the  whole  of  the  hydrochloric  acid  may  be  combined 
with  albumin,  leaving  some  of  the  latter  in  excess  even,  yet  primary 
and  secondary  albumoses  will  appear,  leaving  the  remaining  native 
albumin  to  begin  the  reaction  later.  In  other  words,  it  is  not  neces- 
sary that  one  stage  of  the  digestive  process  must  be  complete  before 
the  following  may  begin.  All  these  reactions  may  be  in  progress 
simultaneously,  and  if  needed  hydrochloric  acid  will  be  taken  from  the 
advanced  products  to  hasten  the  beginning  hydrolysis  of  the  protein 
yet  to  be  digested.  It  has  in  fact  been  shown  that  hydrochloric  acid 
in  combination  with  leucine  and  other  amino  acids,  which  it  will  be 


THE  GASTRIC  JUICE  AND  CHANGES  IN   THE  STOMACH.  I  29 

recalled  are  advanced  products  of  proteolysis,  will  still  digest  fresh 
albumin  rather  rapidly,  but  not  as  well,  of  course,  as  the  equivalent 
amount  of  free  acid. 

The  amount  of  acid  taken  up  by  an  original  native  protein  substance 
during  gastric  digestion  has  been  referred  to  already.  Starting  with 
a  given  weight  of  pure  protein,  hydrochloric  acid  may  be  added  until 
a  distinct  reaction  is  shown  by  dimethylaminoazobenzene.  This  indi- 
cator behaves  as  a  very  weak  base  and  will  show  no  free  acid  until  the 
protein,  considered  as  a  basic  body,  is  saturated.  As  digestion  pro- 
ceeds more  and  more  acid  must  be  added  to  complete  the  saturation. 
The  amount  of  acid  which  may  be  so  added  is  to  some  extent  a  measure 
of  the  advancing  cleavage.  With  phenol-phthalein,  which  is  a  very 
weak  acid,  the  whole  of  the  hydrochloric  acid  behaves  as  "  free  "  acid. 
The  acid  joined  to  the  protein  is  "combined"  acid  as  far  as  the 
dimethylaminoazobenzene  is  concerned  and  this  indicator  may  be  used 
to  show  the  excess  of  free  acid  in  examinations  of  stomach  contents. 
More  will  be  said  about  this  below. 

As  hydrolytic  digestion  goes  on  the  amount  of  water  combined 
becomes  appreciable,  and  finally  may  reach  three  or  four  per  cent,  as 
has  been  determined  by  direct  experiment.  In  a  series  of  investiga- 
tions carried  out  in  the  author's  laboratory  with  casein  the  water  addi- 
tion amounted  to  3.6  per  cent,  and  the  acid  addition,  at  the  same  time, 
to  J.2  per  cent.  The  water  and  acid  are  added  in  molecular  propor- 
tions, therefore.  The  analysis  of  the  albumose  and  peptone  products 
shows  practically  the  same  thing;  these  substances  are  always  lower  in 
carbon  than  are  the  original  proteins  since  oxygen  and  hydrogen  have 
been  taken  up  in  the  cleavage.  These  products  of  diminished  molec- 
ular weight  pass  from  the  stomach  in  the  condition  of  hydrochloride 
salts  into  the  small  intestine,  where  they  undergo  a  new  order  of 
changes. 

THE  ISOLATION  OF  PEPSIN. 

It  has  been  stated  already  that  not  one  of  the  enzymes  is  known  in 
even  approximately  pure  condition.  Very  strong  active  extracts  of 
the  secretion  of  the  gastric  glands  of  animals  may  be  made  by  the  use 
of  various  solvents.  Such  extracts  naturally  contain  much  besides  the 
pepsin,  but  they  are  suitable  for  experimental  and  other  purposes.  A 
good  process  originally  suggested  by  Wittich  is  illustrated  by  the  fol- 
lowing experiment. 

Experiment.     Separate  the  fresh  mucous  membrane  of  the  hog's  stomach  from 
the  outer  coatings   and   mince   it   fine   in    a  meat   chopping  machine.     To   10  gm. 
of  the  minced  membrane  add  200  cc.  of  glycerol  to  which  a  little  hydrochloric  acid 
10 


130  PHYSIOLOGICAL    CHEMISTRY. 

has  been  added.  The  acid  should  amount  to  about  o.i  per  cent  of  the  weight  of 
the  glycerol,  and  may  be  added  in  the  form  of  the  "  normal "  volumetric  acid 
of  which  5  cc.  will  be  sufficient.  Allow  the  mixture  to  stand  a  week  with  frequent 
shaking,  then  filter  it  by  aid  of  the  pump.  This  extract,  bottled,  will  keep  many 
months.  For  use  5  cc.  of  it  may  be  diluted  with  ioo  cc.  of  water  containing  the 
right  amount  of  hydrochloric  acid,  generally  o.i  to  0.3  per  cent. 

For  many  laboratory  experiments  a  fresh  aqueous  extract  is  prefer- 
able which  may  be  secured  in  this  manner : 

Experiment.  The  washed  mucous  membrane  of  the  hog's  stomach  is  chopped 
fine  and  then  rubbed  up  in  a  mortar  with  sharp  sand  or  powdered  glass.  Water 
is  added  (plus  0.1  per  cent  HC1)  in  amount  ten  times  as  great  as  the  weight  of  the 
minced  membrane,  the  mixture  is  thoroughly  stirred,  and  is  allowed  to  stand  over 
night.     It  is  then  filtered  and  is  ready  for  use.     Such  an  extract  is  relatively  strong. 

A  much  purer  product  may  be  secured  by  the  following  process  as 
worked  out  by  Kuehne  and  Chittenden : 

Experiment.  Remove  the  mucous  membrane  of  a  hog's  stomach,  wash  it  thor- 
oughly with  water  and  spread  it  out  on  a  plate  of  glass.  Scrape  the  membrane 
with  a  knife  or  piece  of  glass  and  mix  the  scrapings  with  hydrochloric  acid  of  0.2 
per  cent  strength.  About  half  the  membrane  should  be  reduced  to  the  form  of 
scrapings  and  for  this  mass  500  cc.  of  the  acid  may  be  used.  Allow  this  to  digest 
at  a  temperature  of  400  C.  for  about  two  weeks  in  order  to  convert  as  much  as 
possible  of  the  protein  present  into  peptone.  The  mixture  is  filtered,  and  to  the 
filtrate  powdered  ammonium  sulphate  is  added  to  complete  saturation.  The  object 
of  this  is  to  throw  down  the  pepsin  and  some  albumose,  the  peptone  formed  in 
the  digestion  being  left  in  solution.  This  precipitate  is  collected  on  a  filter,  washed 
with  saturated  ammonium  sulphate  solution  and  redissolved  in  a  little  0.2  per  cent 
hydrochloric  acid.  The  solution  so  obtained  is  placed  in  a  tube  dialyzer  with  a 
little  thymol  water  and  dialyzed  in  running  water  until  the  sulphate  is  all  removed. 
The  pepsin  solution  left  is  mixed  with  an  equal  volume  of  0.4  per  cent  hydro- 
chloric acid  and  kept  at  400  C.  5  days  to  complete  peptonization  of  albumose  still 
present.  Then  precipitation  with  ammonium  sulphate  to  saturation  is  again  effected, 
the  precipitate  collected  and  washed  as  before  and  taken  up  with  0.2  per  cent 
hydrochloric  acid.  This  solution  is  dialyzed  in  running  water  for  the  removal  of 
all  sulphate.  The  liquid  remaining  in  the  dialyzer  is  a  comparatively  pure  pepsin 
solution.  It  may  be  concentrated  in  shallow  dishes  or  on  glass  plafes  at  a  tem- 
perature not  above  400  C,  and  leaves  finally  a  scale  residue.  It  may  be  evaporated 
perhaps  better  in  shallow  dishes  placed  in  a  large  vacuum  desiccator  with  sulphuric 
acid.  The  flakes  or  scales  resulting  may  be  kept  in  dry  form  almost  indefinitely 
and  will  be  found  extremely  active. 

Commercial  Pepsin.  What  is  commonly  known  as  pepsin  is  a 
product  prepared  on  the  large  scale  from  the  hog's  stomach  and  pre- 
served in  dry  form.  Sometimes  the  mucous  membrane  is  cut  into 
shreds,  dried  at  a  low  temperature  and  ground  to  a  powder,  in  which 
condition  it  keeps  very  well.  In  presence  of  weak  hydrochloric  acid 
this  powder  becomes  active  and  is  able  to  digest  a  large  amount  of 
albumin.  Usually,  however,  the  mucous  membrane  is  extracted  in 
some  manner  as  illustrated  by  the  first  steps  described  in  the  Kuehne- 
Chittenden  process.     In  the  commercial  processes  the  following  steps 


THE  GASTRIC  JUICE  AND  CHANGES  IN   THE  STOMACH.  I  3  I 

are  much  simpler  however.  As  carried  out  in  the  United  States,  they 
aim  to  furnish  a  finished  dry  product,  one  part  of  which  will  digest 
3,000  parts  of  egg  albumin  prepared  in  a  certain  way.  Several  dif- 
ferent methods  are  in  use  by  manufacturers  for  purifying  and  concen- 
trating the  extract  from  the  stomach  glands. 

Some  Reactions  with  Pepsin.  The  behavior  of  peptic  extracts 
may  be  easily  shown  by  experiment.  For  this  purpose  an  extract 
made  by  the  use  of  glycerol,  as  described  above,  is  very  convenient. 
An  aqueous  extract  will  answer  if  freshly  prepared. 

Experiment.  Boil  an  egg  until  it  is  hard,  take  out  the  white  portion  and  rub 
it  through  a  clean  wire  sieve  with  fine  meshes,  by  means  of  a  spatula.  Add  about 
five  gm.  of  this  egg  to  100  cc.  of  0.2  per  cent  hydrochloric  acid  in  a  flask,  and  then 
add  2  cc.  of  the  glycerol  extract.  Keep  the  flask  at  a  temperature  of  400  C,  with 
frequent  shaking.  In  time  the  egg  albumin  will  dissolve,  forming  an  opalescent 
liquid.  Unless  the  flask  is  very  frequently  shaken  the  solution  of  the  albumin  will 
be  slow.     Use  the  solution  for  experiment  to  be  described. 

Experiment.  To  2  cc.  of  the  glycerol  extract  in  a  test-tube  add  a  little  water 
and  boil  a  few  minutes.  Now  add  this  boiled  liquid  to  albumin  and  0.2  per  cent 
hydrochloric  acid,  as  in  the  last  experiment,  and  note  that  under  the  same  conditions 
digestion  does  not  take  place,  the  heating  having  destroyed  the  active  enzyme.  In 
like  manner  it  may  be  shown  that  the  enzyme  is  destroyed  by  alkalies  or  stronger 
acids. 

Experiment.  Tests  for  proteoses  and  peptones.  Some  instructive  experiments 
may  be  made  with  the  digesting  mixture  just  described.  Some  hours  after  the 
beginning  of  the  digestion  pour  or  filter  off  as  much  of  the  liquid  as  possible 
and  use  it  in  this  way.  Divide  the  filtrate  into  several  small  portions.  Boil  one 
portion  in  a  test-tube  and  observe  that  it  does  not  coagulate.  On  cooling  the  con- 
tents of  the  tube  the  addition  of  alcohol  produces  a  rather  voluminous  precipitate. 
With  other  small  portions,  a  few  drops  is  enough,  try  the  xantho-proteic  and 
the  Millon's  and  other  color  tests.  These  all  give  good  reactions.  Then  neutra- 
lize the  remainder  of  the  liquid  with  ammonia,  exactly,  and  add  powdered  am- 
monium sulphate  to  saturation.  In  this  way  we  secure  a  precipitate  of  the  proteose 
fraction.  After  a  time  filter  off  this  flocculent  precipitate  and  test  the  filtrate  for 
the  more  advanced  digestion  product,  the  peptone.  The  biuret  reaction  may  be 
employed,  adding  enough  sodium  hydroxide  to  cause  a  separation  of  sodium  sul- 
phate first.  Because  of  the  extreme  solubility  of  the  peptone  bodies  a  real  sepa- 
ration is  extremely  difficult,  but  by  concentration,  and  crystallization  of  the  greater 
part  of  the  ammonium  sulphate  after  cooling,  followed  by  precipitation  by  alcohol, 
it  is  possible  to  secure  a  solution  in  which  the  peptones  may  be  more  clearly 
recognized. 

In  practice  pepsin  is  always  valued  by  the  amount  of  protein  matter 
it  will  digest  in  a  given  time.  Hard-boiled  white  of  egg  is  generally 
employed  with  hydrochloric  acid  of  0.3  per  cent  strength.  Sometimes 
well-washed  fibrin  is  used,  with  a  somewhat  weaker  acid.  As  an  illus- 
tration of  practical  pepsin  testing  the  following  may  be  given,  which  is 
essentially  the  process  of  the  U.  S.  Pharmacopoeia : 

Pepsin  Valuation.    A.    Prepare  AV10  hydrochloric  acid  such  as  is  employed  in 

volumetric  analysis.     B.  Dissolve  66  milligrams  of  pepsin  in  ioo  cc.  of  water.     Mix 


I32  PHYSIOLOGICAL    CHEMISTRY. 

175  cc.  of  A  with  25  cc.  of  B,  giving  a  solution  of  about  0.32  per  cent  acid  strength. 

Boil  a  fresh  hen's  egg  fifteen  minutes,  then  cool  it  by  placing  in  cold  water. 
Separate  the  coagulated  white  part  and  rub  it  through  a  clean  sieve  having  40 
meshes  to  the  linear  inch.  Reject  the  first  portions  which  pass  through.  Weigh 
out  exactly  10  gm.  of  the  clean  disintegrated  substance,  place  it  in  a  100  cc.  flask 
and  add  40  cc.  of  the  acid-pepsin  mixture  last  described.  Put  the  flask  in  a  large 
water-bath  or  thermostat  kept  at  50°  C.  and  let  it  remain  three  hours,  shaking 
gently  every  fifteen  minutes.  At  the  end  of  this  time  the  albumin  should  have 
practically  disappeared,  leaving  at  most  only  a  few  insoluble  flakes.  Much  de- 
pends on  keeping  the  temperature  constant,  and  shaking  at  regular  intervals. 

In  the  above  test  if  the  albumin  is  all  digested  it  shows  that  the  pepsin  has  a 
converting  power  of  3,000  or  over,  which  meets  the  practical  requirement  of  the 
Pharmacopoeia.  The  relative  digesting  power  of  stronger  or  weaker  pepsin  may  be 
ascertained  by  finding  through  repeated  trials  how  much  of  a  pepsin  solution  mixed 
with  the  acid  and  made  up  to  40  cc.  will  be  required  to  dissolve  the  10  gm.  of 
disintegrated  white  of  egg  under  the  same  conditions.  The  process,  although  not 
thoroughly  satisfactory,  is  a  good  one  for  practical  purposes. 

THE  EXAMINATION  OF  STOMACH  CONTENTS. 

From  the  clinical  standpoint  the  examination  of  the  contents  of  the 
stomach  at  any  given  time  is  a  matter  of  considerable  importance. 
The  examination  may  extend  to  the  detection  or  recognition  of  the 
nature  of  various  solid  products  present,  but  ordinarily  it  is  confined 
to  the  detection  or  estimation  of  the  acid  and  the  enzymes  on  which 
the  functional  activity  of  the  organ  depends.  For  such  examinations 
it  is  necessary  to  collect  the  liquid  contents  of  the  stomach  by  the  aid 
of  some  kind  of  stomach  tube.  Vomited  matter  may  be  used  for  the 
same  tests.  In  any  event  it  is  preferable  to  have  as  much  of  the  solid 
contents  as  possible  along  with  the  liquid. 

Inasmuch  as  the  secretion  of  the  gastric  juice  does  not  take  place  all 
the  time,  as  was  pointed  out  above,  but  depends  largely  on  the  action 
of  certain  stimuli,  of  which  the  passage  of  food  down  into  the  stomach 
is  the  common  and  most  important  one,  it  is  customary  to  encourage 
the  flow  of  the  secretion  by  giving  what  is  called  a  "  test-meal "  some 
time  before  introducing  the  stomach  tube.  Unless  this  is  done  it  might 
be  possible  to  collect  from  the  stomach  a  liquid  practically  free  from 
either  acid  or  enzyme.  The  Ewald  test-meal  consists  of  wheat  bread 
and  water  or  tea  without  sugar;  50  gm.  of  bread  to  400  cc.  of  water 
is  an  average  meal.  The  content  of  protein  in  this  would  amount  to 
less  than  5  gm.  ordinarily,  and  in  the  normal  stomach  enough  hydro- 
chloric acid  to  more  than  combine  with  this  would  soon  be  secreted. 
After  about  an  hour  therefore  "  free  "  acid  should  be  detected  by  the 
tests  given  below.  With  a  meal  richer  in  proteins  more  time  would 
be  consumed  in  producing  an  excess  of  hydrochloric  acid.  In  such  a 
case  two  or  three  hours  might  elapse  before  it  would  be  possible  to 


THE  GASTRIC  JUICE  AND  CHANGES  IN   THE  STOMACH.  I  33 

detect  the  free  acid.  The  Riegel  test-meal  consists  of  a  plate  of  broth 
or  soup,  200  gm.  of  beefsteak,  50  gm.  of  wheat  bread  and  200  cc.  of 
water.  The  protein  in  this  would  amount  to  about  60  gm.,  which 
would  require  2  to  3  gm.  of  hydrochloric  acid  for  preliminary  satura- 
tion. Some  hours  would  therefore  be  consumed  in  producing  this. 
The  detection  of  free  acid,  then,  in  such  a  case  would  be  evidence  of 
relatively  high  secreting  power. 

The  Detection  of  Free  Acid.  In  the  early  digestion  stages  of  a 
meal  rich  in  carbohydrates  organic  acids,  especially  lactic  acid,  may  be 
formed  by  bacterial  fermentation.  But  the  amount  so  produced  is 
usually  very  small  if  the  normal  secretion  of  hydrochloric  acid  begins 
in  the  proper  time.  The  organic  acids  produced  are  in  amounts  ordi- 
narily below  0.1  per  cent.  Pathologically,  when  the  bacterial  fer- 
mentation goes  on  unchecked  by  the  production  of  hydrochloric  acid, 
the  organic  acid  may  accumulate  far  beyond  this  and  may  then  be 
readily  detected  by  the  processes  given  below.  At  present  the  detec- 
tion of  the  free  hydrochloric  acid  will  be  considered.  Some  of  the 
gastric  secretion  collected  by  a  tube  or  otherwise  is  filtered,  and  the 
filter  (always  a  small  one)  is  washed  with  a  very  little  water.  The 
mixed  filtrate  and  washings  is  used  for  the  following  tests : 

Dimethylaminoazobenzene  Test.  To  a  few  cc.  of  the  gastric  filtrate  add  a 
drop  or  two  of  this  reagent  used  in  weak  alcoholic  solution  (about  0.2  per  cent). 
Free  hydrochloric  acid  present  strikes  a  pink  or  even  red  color  with  the  indicator. 
Combined  acid  and  the  traces  of  organic  acids  which  may  be  present  have  no  such 
action. 

Congo  Red  Test.  This  substance  in  aqueous  solution  is  turned  blue  by  very 
dilute  hydrochloric  acid.  Organic  acids  do  not  give  the  test,  except  when  present 
in  relatively  much  stronger  solution. 

The  reaction  is  most  conveniently  carried  out  by  means  of  test  papers  made  by 
dipping  filter  paper  in  a  solution  of  the  coloring  matter  and  drying.  These  strips 
are  dipped  in  the  gastric  filtrate  and  allowed  to  dry  spontaneously. 

Methyl- Violet  Test.  A  dilute  violet-colored  aqueous  solution  of  this  substance, 
when  mixed  with  weak  hydrochloric  acid,  turns  blue.  The  reaction  with  gastric 
juice  is  faint,  but  when  care  is  observed,  characteristic.  Organic  acids,  even  when 
present  in  quantity,  do  not  give  the  test,  which  was  first  successfully  used  for  the 
detection  of  traces  of  mineral  acids  in  vinegar.  Use  a  few  drops  with  2  cc.  of  the 
gastric  filtrate. 

Guenzberg's  REAGENT.     This  is  a  well-known  solution  and  is  made  as  follows: 

Phloroglucin   2  grams 

Vanillin    1  gram 

ihol    100  cubic  centimeters 

To  make  the  tesl  for  free  hydrochloric  acid,  mix  5  cc.  of  this  solution  with 
5  cc.  of  the  gastric  filtrate  and  concentrate  in  a  u'ass  or  porcelain  vessel  on  the 
water-bath.  In  presence  of  free  hydrochloric  acid  the  liquid  gradually  becomes 
red  as  the  concentration  proo 


134  PHYSIOLOGICAL    CHEMISTRY. 

Boas'  Reagent  for  Free  Hydrochloric  Acid. 

Resorcinol   5  grams 

Cane  sugar  3  grams 

Alcohol,  50  per  cent ioo  grams 

Add  a  few  drops  and  evaporate  as  above.     Color  appears  as  in  the  other  test. 

Total  Hydrochloric  Acid.  By  the  use  of  the  above  tests  the  excess 
of  hydrochloric  acid  beyond  that  which  the  proteins  and  bases  will  hold 
is  recognized.  At  one  time  this  acid  was  supposed  to  be  all  that  could 
have  any  physiological  value.  The  importance  of  that  combined  with 
the  proteins  in  the  form  of  acid  albumin  was  not  considered.  From 
the  explanations  given  above  it  is  evident  that  in  some  stages  of  the 
digestive  process  the  hydrochloric  acid  may  be  largely  or  wholly  in 
combination  and  therefore  not  in  a  form  to  be  recognized  through  the 
aid  of  the  tests  just  given.  From  experiments  made  under  such  con- 
ditions it  would  be  wrong  to  conclude  that  the  stomach  is  secreting  no 
acids.  It  has  been  found  that  by  making  the  test  in  a  different  way, 
employing  phenol-phthalein  instead  of  the  reagents  mentioned  above, 
the  combined  acid  may  be  readily  recognized.  To  do  this  we  must 
make  practically  a  quantitative  analysis,  and  the  method  employed 
depends  on  the  proper  use  of  certain  indicators.  This  will  be  taken 
up  presently. 

The  Organic  Acids.  Under  normal  conditions,  as  already  stated, 
these  are  present  in  the  stomach  contents  in  very  small  amounts  only. 
As  their  formation  depends  on  bacterial  fermentation  processes,  they 
appear  only  when  hydrochloric  acid  is  absent,  or  present  in  relatively 
small  proportion.  Mineral  acids  arrest  bacterial  fermentation  quickly, 
from  which  it  follows  that  in  the  healthy  stomach  there  is  never  oppor- 
tunity for  the  accumulation  of  much  lactic  or  other  acid  of  like  origin. 
These  acids  are  never  products  of  secretion  as  is  hydrochloric  acid; 
they  are  not  formed  in  the  cells  of  the  walls  of  the  stomach,  but  in  the 
food  contents.  If  from  some  pathological  cause  the  fundus  glands 
fail  to  secrete  hydrochloric  acid  or  secrete  it  in  traces  only,  then  the 
fermentation  bacteria  can  work  unhindered  on  the  carbohydrates  in 
the  stomach  and  produce  relatively  large  amounts  of  acid.  Lactic 
acid  is  usually  the  most  abundant  of  these  fermentation  products,  but 
butyric  acid  is  occasionally  formed  and  also  acetic  acid. 

The  recognition  of  these  organic  acids  is  not  difficult  if  they  are 

alone  or  mixed  with  only  a  little  mineral  acid.     These  are  of  course 

the  cases  of  practical  importance;  much  hydrochloric  acid  and  much 

lactic  acid  could  not  be  found  together.     Among  the  simpler  reactions 

•  employed  the  following  with  iron  salts  are  the  most  useful. 


THE  GASTRIC  JUICE  AND  CHANGES  IN   THE  STOMACH.  135 

Test  for  Lactic  Acid.  Prepare  a  dilute  solution  of  phenol  by  dissolving  I  gm. 
of  the  pure  crystallized  product  in  75  cc.  of  water.  To  this  add  5  drops  of  a  strong 
solution  of  ferric  chloride,  which  produces  a  deep  blue  color.  Five  cc.  of  this  mix- 
ture suffices  for  a  test.  Add  to  it  a  few  drops  of  the  liquid  containing  lactic  acid, 
and  note  the  change  from  blue  to  yellow.     (Uffelmann's  test.) 

A  weak,  colorless  solution  of  ferric  chloride  serves  also  as  a  test  substance,  as  its 
color  becomes  much  deeper  by  addition  of  a  trace  of  lactic  acid.     (Kelling's  test.) 

This  reaction  is  not  influenced  by  the  presence  of  small  amounts  of  hydrochloric 
acid,  as  can  be  readily  shown  by  adding  some  to  the  liquid  to  be  tested.  The  color 
change  depends  on  the  peculiar  behavior  of  ferric  salts  with  organic  acids  in  general. 
These  acids  are  relatively  weak  and  with  ferric  iron  tend  to  form  "  undissociated  " 
salts  which  all  have  a  deeper  color  than  have  those  with  the  stronger  acids. 

Both  of  these  tests  are  much  more  delicate  if  applied  to  the  product  obtained  by 
shaking  out  the  gastric  juice  or  stomach  content's  with  ether.  About  10  cc.  of  the 
filtered  juice  may  be  shaken  with  100  cc.  of  ether  in  a  separatory  funnel  through 
half  an  hour.  When  the  ether  is  drawn  off  and  evaporated  slowly  the  lactic  acid, 
if  present,  is  left  as  a  residue.  This  residue  is  taken  up  with  a  few  cc.  of  water 
and  used  for  the  tests. 

The  Amount  of  Acid.  It  has  just  been  shown  how  we  are  able  to 
recognize  the  free  acids  existing  in  the  gastric  juice,  and  also,  under 
certain  conditions,  that  in  combination  with  the  protein.  An  equally 
important  problem  is  the  determination  of  the  proportions  in  which 
these  fractions  of  the  total  acid  exist.  Several  different  schemes  have 
been  proposed  by  which  these  degrees  of  acidity  may  be  measured. 
The  total  acid  not  combined  in  the  form  of  inorganic  salts  may  be  most 
accurately  found  by  the  methods  of  ordinary  quantitative  analysis. 
The  total  chlorine  is  found  by  precipitation  or  by  the  Volhard  titration. 
The  total  bases  are  found  by  the  usual  gravimetric  methods.  On  cal- 
culating the  amount  of  chlorine  necessary  to  combine  with  these  bases 
an  excess  is  left  over  which  must  be  considered  as  existing  in  the  form 
of  free  acid.  In  very  exact  work  the  traces  of  phosphates  and  sul- 
phates present  must  be  also  determined  and  these  first  combined  with 
bases.  The  method  is  one  which  requires  great  care  in  manipulation, 
and  besides  does  not  distinguish  between  free  acid  and  that  held  as 
acid  albumin,  and  this  is  a  very  important  point. 

The  principle  of  another  general  method  may  be  illustrated  in  this  way.  Three 
portions  of  the  gastric  juice  of  5  cc.  each  are  measured  off.  The  first  is  mixed 
with  a  little  pure  sodium  carbonate,  evaporated  and  ignited.  The  total  chlorine  is 
so  retained  and  may  be  found  by  the  Volhard  titration.  The  second  portion  is 
eyaporated  slowly  to  dryness  at  a  low  temperature,  mixed  with  sodium  carbonate 
and  ignited.  The  chlorine  is  determined  in  the  residue.  This  represents  the 
fractions  combined  to  proteins  and  to  inorganic  bases,  as  the  free  hydrochloric  acid 
t  in  the  original  evaporation  at  low  temperature.  Finally,  the  third  portion 
is  evaporated  and  ignited  without  any  addition.  The  chlorine  now  found  in  the 
residue  is  that  which  originally  existed  in  inorganic  combination.  With  these 
three  operations,  as  is  at  once  apparent,  it  is  possible  to  measure  the  element  in 
the  three  kinds  of  combination.  The  process  has  been  modified  and  improved  so 
as  to  be  fairly  exact. 


I36  PHYSIOLOGICAL    CHEMISTRY. 

Attempts  are  now  made  to  determine  the  acid  accurately  volumet- 
rically  by  the  aid  of  indicators,  and  here,  it  may  be  said,  if  we  can 
neglect  the  lactic  acid  present,  pretty  good  results  are  possible.  But 
if  the  lactic  acid  is  present  in  amount  more  than  traces,  as  suggested 
by  the  qualitative  tests  above,  the  process  becomes  more  difficult. 
Before  describing  the  details  of  a  method  something  must  be  said 
about  the  indicators  themselves,  as  an  understanding  of  their  nature 
and  behavior  is  necessary  for  much  that  is  to  follow. 

Theory  of  Indicators.  The  indicators  employed  in  acidimetry  and  alkalimetry  are 
all  weak  acids  or  weak  bases  themselves,  and  in  general  much  weaker  than  the 
acids  or  bases  in  the  determination  of  which  they  are  employed.  These  indicators, 
as  acids  or  bases,  form  salts  with  the  bases  or  acids  to  be  titrated;  it  is  on  the 
peculiar  properties  of  these  salts  that  the  value  of  the  indicators  depends.  As  is 
well  known  the  change  in  "  reaction  "  in  employing  an  indicator  is  accompanied  by 
a  change  in  color.  This  change  in  color  is  accounted  for  in  two  general  ways. 
According  to  one  view,  which  is  usually  described  as  the  "  chromophoric  theory " 
substances  which  may  be  employed  as  indicators  must  be  capable  of  existing  in 
fwo  modifications,  one  of  which,  at  least,  must  possess  a  so-called  chromophoric 
group.  By  change  of  reaction  one  of  these  modifications  must  pass  over  into  the 
other  practically  instantaneously,  and  by  the  addition  of  the  smallest  excess  of 
alkali  or  acid.  Hundreds  of  substances  show  this  phenomenon  in  a  general  way, 
but  to  be  of  use  as  indicators  the  change  must  be  both  rapid  and  delicate. 

Phenol-phthalein,  for  example,  may  be  assumed  to  exist  in  two  forms,  one  of 
which  is  an  extremely  weak  carboxylic  acid,  and  weaker  than  the  acids  which  are 
to  be  titrated  by  its  aid.  The  acid  itself  is  not  stable,  but  it  forms  more  stable 
red  salts  with  alkalies.  By  addition  of  acids  the  phenol-phthalein  passes  over  into 
the  other  form,  which  is  a  lactone  and  colorless.  The  value  of  the  indicator 
depends  on  the  fact  that  these  changes  are  extremely  sharp.  The  salt  form  has 
a  chromophoric  complex  which  appears  to  be  of  quinoid  structure. 

In  methyl  orange,  or  its  related  substances,  we  have  evidently  two  chromophoric 
groups,  one  of  which  is  found  in  the  yellow  salt  form,  given  with  alkalies,  and  the 
other  in  the  red  isomer  produced  when  combined  with  acids.  The  stable  yellow 
form  is  produced  by  even  very  weak  alkalies,  while  the  weakest  acids  are  not  able 
to  effect  a  transformation  into  the  red  isomer.  This  property  has  its  advantages, 
in  the  titration  of  mixtures  containing  both  strong  and  weak  acids. 

The  other,  and  perhaps  more  commonly  accepted,  view  of  indicators  is  based 
on  the  ionization  theory.  Phenol-phthalein  is  assumed  to  possess  a  red  ion  which 
does  not  appear  in  the  acid  form  because  of  its  slight  dissociation,  but  when  com- 
bined with  alkalies  the  salt  dissociates  and  the  red  ion  then  shows  itself.  With 
extremely  weak  alkalies  this  change  does  not  follow,  but  the  weakest  acids  are 
able  to  suppress  the  ionization  and  with  it  the  color.  Hence  the  value  of  the 
indicator  in  titrating  weak  organic  acids.  As  weak  bases  do  not  form  stable  salts 
with  very  weak  acids,  the  whole  of  the  hydrochloric  acid  combined  with  protein 
may  be  titrated,  as  illustrated  by  this  equation : 

Prot.  HC1  +  NaOH  =  Prot.  +  NaCl  +  H20. 

This  reaction  will  be  studied  more  fully  later. 

Methyl  orange  exhibits  the  opposite  behavior,  and  is  assumed  to  act  as  a  weak 
base  which  in  the  undissociated  form  is  yellow.  The  ion  of  the  salts,  formed  with 
acids,  is  red.  With  weak  acids  it  forms  extremely  unstable  salts  and  therefore 
cannot  be  used  in  the  titration  of  such  acids.     Carbonic  acid  is  practically  inert 


THE  GASTRIC  JUICE  AND  CHANGES  IN  THE  STOMACH.  137 

with  it.  But  bases,  even  very  weak  ones,  are  able  to  displace  it  from  its  combina- 
tions with  acids,  just  as  weak  acids  displace  phenol-phthalein.  Weak  ammonia, 
for  example,  which  combines  imperfectly  with  phenol-phthalein,  is  strong  enough 
to  react  with  the  acid  combinations  of  the  dimethylaminoazobenzene  or  methyl 
orange.  Protein  in  the  so-called  acid  albumin  combination,  in  which  the  protein 
is  really  basic  in  character,  is  stronger  than  the  indicator  and  able  to  displace  it 
from  it's  salts.  If  we  add  a  weak  alkali  to  a  solution  of  the  red  salt  of  methyl 
orange  the  color  changes  immediately  on  the  neutralization  of  any  free  acid  which 
may  be  present.  The  yellow  color  of  the  undissociated  base  takes  the  place  of  the 
red  of  the  salt  or  free  ion.  With  the  very  weak  solutions  of  the  indicator  used 
the  merest  drop  of  alkali  should  be  sufficient  to  bring  about  the  change  in  the 
indicator  salt  alone.  Assuming  in  solution  a  mixture  of  free  hydrochloric  acid, 
protein  and  hydrochloric  acid,  and  the  red  methyl  orange-hydrochloric  acid  salt, 
addition  of  weak  sodium  hydroxide  would  produce  a  change  in  color  immediately 
after  the  neutralization  of  the  last  trace  of  free  hydrochloric  acid.  Any  excess  of 
alkali  added  would  separate  the  protein-acid  combination,  but  the  protein  would 
behave  itself  as  a  base  and  furnish  hydroxyl  ions  to  decrease  the  dissociation  of 
the  indicator  and  produce  the  characteristic  yellow.  Hence  the  "  neutral "  point 
is  reached  with  the  disappearance  of  the  actually  uncombined  HC1.  With  a  weak 
acid,  like  lactic  acid,  present  in  small  amount  the  condition  would  be  practically  the 
same.  Such  an  acid  is  but  slightly  ionized  and  not  able  to  form  stable  salts  with 
the  indicator. 

Illustration.  Before  taking  up  the  actual  titration  of  the  stomach  contents  a 
practical  illustration  of  the  steps  may  be  found  useful.  To  this  end  a  mixture  of 
about  10  gm.  of  finely  divided  coagulated  egg  albumin  with  10  milligrams  of 
powdered  pepsin  and  ioo  cc.  of  0.4  per  cent  hydrochloric  acid  should  be  made 
up  to  200  cc.  with  water.  This  will  give  an  acid  strength  at  the  very  outset  of 
0.2  per  cent. 

Immediately  after  diluting  measure  out  three  portions  of  25  cc.  each  of  the 
thoroughly  shaken  mixture.  Filter  one  portion  (A)  at  once  and  wash  the  residue 
on  the  filter  with  water  several  times,  adding  the  washings  to  the  filtrate.  Add  a 
few  drops  of  phenol-phthalein  solution  and  titrate  this  liquid  with  AVioNaOH, 
preferably  after  warming.  Warm  the  second  portion  (B)  of  25  cc.  and  titrate  with 
the  alkali  and  phenol-phthalein  without  filtering.  In  general  the  result  here  will  be 
higher  than  in  the  first  case.  It  represents  the  total  acidity  and  corresponds  to 
one-eighth  of  the  acid  taken.  In  the  titration  of  A  the  result  will  be  lower  because 
a  part  of  the  acid  combined  at  once  with  the  albumin  and  is  left  in  a  form  not  yet 
soluble.  To  the  third  portion  (C)  of  25  cc.  measured  off  add  2  drops  of  a  weak 
dimethylaminoazobenzene  indicator  and  titrate  directly  with  the  N/10  alkali.  The 
result  will  be  found  distinctly  lower  than  that  with  A  or  B,  even  in  this  beginning 
stage  of  the  process. 

The  remainder  of  the  albumin  and  acid  mixture  in  a  loosely  stoppered  flask  is 
placed  in  a  water-bath  and  kept  as  exactly  as  possible  at  a  temperature  of  400  C. 
through  five  or  six  hours.  The  mixture  is  shaken  frequently  as  in  the  pepsin  test 
described  above.  At  the  end  of  two  or  three  hours  measure  out  two  portions  of 
25  cc.  each  ;  titrate  one  with  phenol-phthalein  addition  and  the  other  with  dimethyl- 
aminoazobenzene. The  result  with  phenol-phthalein  present  should  be  nearly  the 
same  as  before,  while  with  the  other  indicator  it  will  probably  be  a  little  less 
than  in  the  first  case  and  not  much  more  than  half  the  acidity  shown  by  the  phenol- 
phthahin.  After  the  digestion  lias  continued  six  hours,  or  until  practically  com- 
plete,  te»1  two  further  portions  of  25  cc.  each  in  the  same  way.  The  total  acidity 
as  measured  by  the  aid  of  phenol-phthalein  will  be  found  but  slightly  changed, 
while  with  the  dimethylaminoazobenzene  the  "free"  acid  should  be  found  still 
further  lowered  probably,  and  not   over  half  the  total  acid.     The  exact  relation  of 


I38  PHYSIOLOGICAL    CHEMISTRY. 

the  free  to  the  total  acidity  depends  on  the  strength  of  the  pepsin  and  the  amount 
of  albumin  taken.  In  a  long-continued  artificial  digestion  or  in  presence  of  much 
pepsin  the  acid  is  gradually  combined  more  and  more  completely  because  the 
basic  digestion  products  formed  have  relatively  lower  molecular  weights  and  com- 
bine with  the  acid  more  or  less  perfectly,  and  as  shown  below  the  total  acidity  as 
measured  by  phenol-phthalein  will  be  increased.  Exact  numerical  relations  here 
have  not  yet  been  established  by  sufficiently  numerous  or  detailed  experiments. 

Titration  of  the  Gastric  Juice.  The  illustration  given  above  shows 
about  how  this  should  be  carried  out.  In  general  as  large  a  volume 
as  there  used  will  not  be  available,  but  5  or  10  cc.  should  be  collected 
by  the  tube  or  otherwise  for  each  test.  In  testing  for  the  total  acidity 
the  mixture  should  not  be  filtered,  unless  the  digestion  is  far  advanced, 
for  the  reason  just  pointed  out.  A  part  of  the  hydrochloric  acid  may 
be  held  in  the  insoluble  residue.  In  testing  for  the  free  acid,  however, 
the  measured  portion  should  be  filtered  and  the  residue  on  the  filter 
washed  with  a  little  water.  The  whole  of  the  free  acid  will  then  be 
found  in  the  filtrate.  As  the  color  change  with  the  indicator  here  used 
is  not  as  sharp  as  in  the  other  case  a  clearer  liquid  is  essential  for 
the  test. 

These  two  titrations  give  us  the  total  acidity  and  the  free  hydro- 
chloric acid,  but  do  not  measure  the  organic  acid  which  may  possibly 
be  present.  Attempts  have  been  made  to  estimate  this  by  aid  of 
another  indicator.  Sodium  alizarin  sulphonate  has  been  used  for  this 
purpose,  but  the  reaction  is  not  as  sharp  as  desirable.  This  substance 
appears  to  behave  as  a  weak  acid,  but  one  not  as  weak  as  phenol- 
phthalein.  Lactic  acid  may  be  titrated  with  it,  but  protein  separated 
from  HC1  behaves  as  a  base  toward  it.  Theoretically  the  three  indi- 
cators are  related  in  this  way,  as  illustrated  by  diagrams,  in  which 
H  Pht  represents  phenol-phthalein,  HA1  alizarin  sodium  sulphonate, 
Or  CI  the  hydrochloric  acid  salt  of  dimethylaminoazobenzene  and  HL 
lactic  acid : 

H  Pht  1  HA1  ■)  Or  CI  ^ 

HClProt.      L  +  Na0H.  HClProt.     L  +  Na0R  HClProt.     I  +  Na0H. 

HC1  J  HC1  J  HC1  J 

1  2  3 

It  has  been  shown  above  how  phenol-phthalein  and  the  methyl  orange 
bodies  act.  The  alizarin  sulphonate  as  standing  midway  between 
them  in  properties  is  influenced  by  the  protein  which  may  be  separated 
from  acid  albumin  in  titration  with  NaOH.  Therefore  the  difference 
between  the  titrations  in  schemes  numbers  2  and  3  must  measure  the 
lactic  or  similarly  acting  organic  acid. 

Under  some  conditions  this  appears  to  be  true.     When  there  is  rela- 


THE  GASTRIC  JUICE  AND  CHANGES  IN  THE  STOMACH.  139 

tively  much  lactic  acid  present  and  not  much  of  the  digestion  products 
a  fairly  good  end  reaction  is  obtained.  This  corresponds  of  course  to 
a  practical  case  and  the  indicator  then  has  some  value.  But  as  diges- 
tion goes  on  the  products  formed  are  more  or  less  basic.  While  not 
strong  enough  to  affect  the  phenol-phthalein  they  do  appear  to  act  on 
the  alizarin  compound  in  such  a  manner  as  to  diminish  the  alkali 
required  for  titration ;  the  free  hydrochloric  acid  is  thus  made  to  appear 
low.     It  is  plain  that  the  indicator  has  but  limited  value. 

The  Amount  of  Pepsin.  Thus  far  the  detection  and  estimation  of 
the  acid  in  the  stomach  contents  has  alone  been  considered,  but  the 
question  of  the  amount  of  pepsin  present  may  be  of  equal  importance. 
We  have  no  very  satisfactory  tests  to  determine  this  amount,  but 
approximate  values  may  be  obtained  by  observing  the  action  of  a 
filtered  portion  of  the  gastric  juice  on  some  albumin  solution  to  which 
weak  hydrochloric  acid  has  been  added.  Comparative  tests  may  be 
made  in  this  manner : 

Prepare  some  egg  albumin  solution  of  about  2  per  cent  strength  (2  per  cent  of 
dry  albumin)  and  mix  this  with  0.4  per  cent  hydrochloric  acid  in  equal  propor- 
tions ;  that  is  for  every  cubic  centimeter  of  the  albumin  solution  take  one  cubic 
centimeter  of  the  acid  solution.  The  resultant  mixture  has  an  acid  strength  of 
0.2  per  cent  and  an  albumin  strength  of  1.0  per  cent.  Measure  out  20  cc.  of  the 
acid-albumin  mixture  and  add  to  it  5  cc.  of  the  filtered  gastric  juice  or  stomach 
contents.  To  another  20  cc.  of  the  mixture  add  5  cc.  of  a  0.2  per  cent  pepsin 
solution.  Incubate  both  mixtures  through  24  hours  at  a  temperature  of  400  C, 
with  frequent  shaking.  At  the  end  of  the  time  examine  both  the  incubated  liquids 
for  digestion  products.  To  this  end  neutralize  a  few  cc.  of  each  portion  with 
very  weak  alkali,  using  pbenol-phthalein,  and  observe  whether  a  precipitate  forms 
or  not,  directly  or  on  warming  gently.  If  no  precipitate  forms  in  either  fraction 
the  digestion  has  gone  beyond  the  acid  albumin  stage,  which  should  be  the  case 
of  course  in  the  comparison  sample  with  the  pepsin.  Next  test  5  cc.  portions  of 
each  mixture  in  the  Esbach  albuminometer,  adding  the  usual  Esbach  reagent  (10 
gm.  picric  acid  and  20  gm.  citric  acid  with  water  to  1  liter).  This  reagent  precipi- 
tates proteoses  but  not  peptones,  when  used  in  excess,  and  from  the  extent  of  the 
reaction  in  the  tube  some  conclusion  can  be  drawn  as  to  degree  of  digestion.  A 
similar  test  should  be  made  with  potassium  ferrocyanide  and  acetic  acid  in  place 
of  the  picric  and  citric  acids.     Ferrocyanhydric  acid  does  not  precipitate  peptones. 

In  another  general  method  the  action  on  solid  coagulated  protein  is  observed. 
White  of  egg  is  drawn  up  into  narrow  glass  tubes  having  an  internal  diameter  of 
about  2  mm.,  and  coagulated  by  heat.  The  tubes  are  then  cut  into  lengths  of  1 
centimeter,  thus  exposing  the  ends  of  the  coagulated  columns  of  albumin.  These 
prepared  tubes  are  then  immersed  in.  the  filtered  gastric  juice  and  in  standard 
pepsin  solution  to  be  compared  and  kept  at  a  temperature  of  400  some  hours.  The 
change  in  length  of  the  albumin  column  is  taken  as  the  measure  of  the  peptic 
activity.  The  filtered  gastric  juice  must  be  largely  diluted  with  water  before  making 
the  test,  as  salts  and  carbohydrate-  present  interfere  with  the  normal  solution  of 
the  end  of  the  coagulated  mass.  The  amount  of  albumin  dissolved  under  these 
conditions  is  said  to  be  proportional  to  the  square  root  of  the  ferment  strength, 
but  the  rule  is   far   from  exact. 


I4-0  PHYSIOLOGICAL    CHEMISTRY. 

It  has  been  explained  above  that  according  to  late  researches  pepsin 
and  rennin  are  believed  by  many  chemists  to  be  identical  substances. 
As  the  milk  coagulating  behavior  seems  to  be  much  more  easily  fol- 
lowed and  measured  than  the  proteolytic,  the  ferment  strength  is  fre- 
quently determined  by  observing  the  extent  of  the  coagulating  power. 
The  test  may  be  made  in  a  number  of  ways  and  has  already  found 
clinical  application,  but  the  real  value  of  the  process  remains  to  be 
demonstrated. 

PRODUCTS  OF  PEPTIC  DIGESTION. 

Frequent  reference  has  already  been  made  to  the  question  of  what 
is  produced  from  the  food  proteins  during  peptic  digestion.  In  an- 
swering this  question  it  is  necessary  to  distinguish  between  what  may 
be  formed  under  the  influence  of  pepsin  and  hydrochloric  acid,  with 
sufficiently  long  time  afforded  for  the  action,  and  what  actually  is 
formed  in  the  few  hours  in  which  food,  under  normal  conditions, 
remains  in  the  stomach.  On  this  subject  the  views  of  physiological 
chemists  have  undergone  various  and  marked  changes.  The  stomach 
has  long  been  considered,  popularly,  as  the  chief  organ  of  digestion, 
and  this  view  appeared  to  be  confirmed  by  the  results  of  the  earlier 
experiments  carried  out  with  artificial  digestive  mixtures.  The  gradual 
disappearance  of  coagulated  egg  albumin  or  of  fibrin,  with  the  simul- 
taneous formation  of  soluble  products,  is  a  phenomenon  easily  observed. 
Various  precipitation  reactions  served  to  recover  from  the  mixture 
the  products  formed  and  these  were  early  spoken  of  as  "  peptones." 

At  this  time,  however,  the  distinction  between  real  peptones  and 
proteoses  was  not  thought  of.  It  remained  to  be  shown  that  all  this 
abundant  mass  of  material  formed  in  the  course  of  a  few  hours'  diges- 
tion consists  actually  in  the  main  of  products  preliminary  to  peptones. 
This  later  knowledge  came  somewhat  slowly  and  led  to  a  radical  view 
of  just  the  opposite  order  from  the  early  popular  one  of  the  function 
and  importance  of  the  stomach.  If  the  stomach  is  not  the  principal 
organ  of  digestion,  it  was  asked,  what  is  its  real  value?  If  the  opera- 
tions carried  out  there  may  be  accomplished  as  well  later  in  the  intes- 
tine, if  its  work  is  wholly  preliminary  and  if  in  turn  these  preliminary 
stages  are  not  really  essential,  what  are  the  functions  for  which  the 
presence  of  the  stomach  appears  to-be  "practically"  necessary?  A 
number  of  remarkable  experiments  made  with  animals  threw  some 
light  on  the  question.  It  was  found  that  dogs  were  able  to  live  and 
thrive  without  the  stomach,  mixed  foods  of  various  kinds  being  almost, 
if  not  quite,  perfectly  digested  in  the  intestine.  One  of  these  dogs 
was  kept  under  observation  several  years  after  complete  removal  of 


THE  GASTRIC  JUICE  AND  CHANGES  IN  THE  STOMACH.  I4I 

the  stomach,  and  in  other  cases  dogs  have  been  fed  through  long 
periods  by  direct  injection  of  food  into  the  small  intestine,  the  connec- 
tion with  the  stomach  being  meanwhile  completely  broken  by  ligature. 
The  feces  of  these  animals  were  found  to  be  practically  normal  in 
most  cases. 

With  such  facts  in  view  a  school  of  chemists  following  Bunge  have 
come  to  the  conclusion  that  the  main  use  of  the  stomach  is  in  the 
destruction  of  bacteria  taken  in  with  the  food.  The  acid  usually 
present  in  the  gastric  juice  is  assumed  to  be  sufficiently  strong  to 
destroy  most  of  the  ferment  organisms,  which  if  allowed  to  live  and 
pass  into  the  alkaline  intestine  would  certainly  work  great  harm.  It 
must  be  granted  that  this  view  appears  plausible;  the  protection  of  the 
intestine  through  the  sterilizing  action  of  the  acid  is  beyond  question 
of  prime  importance  and  that  the  stomach  actually  accomplishes  this 
to  some  extent  must  not  be  forgotten  in  any  discussion  of  the  relations 
of  the  one  organ  to  the  other.  It  is  well  known  what  happens  in  the 
human  stomach  when,  from  some  cause,  the  hydrochloric  acid  is  tem- 
porarily absent  or  greatly  diminished.  A  great  development  of  organic 
acid-producing  bacteria  follows,  and  the  products  of  these  are  a  source 
of  much  discomfort  without  being  at  the  same  time  strong  enough  to 
cheek  the  growth  of  certain  pathogenic  bacteria. 

But  after  all  these  facts  have  been  given  due  weight  we  must  still 
admit  that  the  peptic  digestion  if  not  actually  "  essential  "  is  in  practice 
really  important.  Some  peptone,  although  not  a  large  amount,  is 
formed  in  the  stomach  and  this  is  ready  for  immediate  absorption,  or 
for  the  further  conversion  by  erepsin.  The  proteoses  are  ready  for 
the  final  conversion  into  peptones,  or  they  may  be  attacked  directly  by 
the  erepsin.  This  preliminary  work  saves,  therefore,  much  work  in 
the  intestine.  In  studying  the  products  of  pancreatic  digestion  it  will 
appear  that  some  of  them  are  identical  with  those  formed  in  the 
stomach,  or  by  pepsin-hydrochloric  acid  action  in  general.  Others 
appear  at  first  sight  quite  distinct  and  their  existence  leads  to  the  long- 
accepted  notion  that  the  peptic  action  is  incapable  of  carrying  the  con- 
version of  proteins  through  to  the  final  stages.  The  conclusions 
which  may  be  drawn  from  the  most  recent  of  the  long  investigations 
which  have  been  carried  out  on  this  question  are  somewhat  conflicting, 
but  in  the  main  they  show  that  with  a  sufficiently  long  time  allowed  the 
end  products  of  peptic  and  tryptic  digestion  are  essentially  the  same. 

Some  idea  of  the  extent  of  the  changes  taking  place  in  reasonably  prolonged 
peptic  digestion  may  be  obtained  from  a  study  of  the  rapidity  of  combination  with 
hydrochloric  acid  which  has  been  already  referred  to  in  speaking  of  digestion 
exp-riments.     A  digestive  mixture  was  made  with  90  gm.  of  coagulated  and  finely 


I42  PHYSIOLOGICAL    CHEMISTRY. 

divided  white  of  egg,  900  cc.  of  approximately  0.2  per  cent  hydrochloric  acid  and 
150  mg.  of  commercial  pepsin.  Two  portions  of  this  mixture,  of  25  cc.  each,  were 
titrated  at  once,  one  with  use  of  phenol-phthalein  and  the  other  with  dimethyl- 
aminoazobenzene.  The  remainder  of  the  mixture  was  poured  into  a  large  flask 
which  was  maintained  at  a  temperature  of  40°  in  a  thermostat  through  a  number 
of  days.  Titrations  were  made  from  time  to  time  with  the  following  results,  25 
cc.  of  the  mixture  being  always  taken.  The  phenol-phthalein  titration  was  made 
warm,  the  other  cold. 

Cc.  oiN/10  Cc.oiN/10 

Time.  NaOH  with  NaOH  with  Dimethyl- 

Phenol-phthalein.  aminoazobenzene. 

at  once  14  9.0 

10  hours  14.5  8.5 

24      "  14-5  8.0 

40      "  14-7  7-5 

06       "  16.0  6.9 

168      "  16.6  6.5 

It  appears,  therefore,  that  the  "  total "  acidity  as  measured  in  the  phenol-phthalein 
titration  undergoes  a  slight  increase.  The  hydrochloric  acid  remains,  and  added 
to  it  are  some  digestive  products  of  amino-acid  character,  and  strong  enough  to 
show  in  this  way.  On  the  other  hand  in  the  course  of  the  week's  digestion  there 
is  a  decrease  in  the  "free"  hydrochloric  acid  as  measured  by  aid  of  the  dimethyl- 
aminoazobenzene  indicator.  The  titration  here  is  not  as  sharp  as  with  phenol- 
phthalein,  but  close  enough  to  indicate  the  facts.  An  amount  of  acid  correspond- 
ing to  9  cc.  of  the  N/10  alkali  was  "  free  "  immediately  after  mixing.  About  5  cc. 
had  evidently  combined  with  the  egg  albumin  to  form  the  acid  albumin.  As 
digestion  progressed,  and  smaller  molecules  were  formed,  more  acid  was  required 
to  unite  with  these.  Finally  the  perfectly  uncombined  acid  amounted  to  the  equi- 
valent of  6.5  cc.  of  alkali  only.  Before  the  end  of  the  digestion  bodies  were  formed 
which  acted  as  both  acids  and  bases  with  the  proper  indicators.  The  amino  acids 
are  of  this  character. 

Some  similar  results  were  obtained  in  the  author's  laboratory  in  a  very  prolonged 
digestion  of  casein  with  pepsin  and  hydrochloric  acid.  A  mixture  was  made 
containing  in  1000  cc.  9.6  grams  of  pure  casein,  2.33  grams  of  hydrochloric  acid 
and  500  milligrams  of  commercial  pepsin.  This  was  incubated  at  380,  and  from 
time  to  time  portions  were  withdrawn  for  titration  with  N/10  NaOH.  The  fol- 
lowing data  were  obtained.  The  original  acid  was  of  such  strength  that  25  cc. 
required  16  cc.  of  the  alkali  for  titration  with  phenol-phthalein  or  methyl  orange. 
The  first  titration  after  mixing  with  the  casein  was  made  at  once. 

Time. 

at  once 
24  hours 
48  hours 
13  days 
29  days 
38  days 
54  days 

As  the  digesting  mixture  leaves  a  residue  of  so-called  pseudo-nuclein  the  titra- 
tion is  not  quite  as  sharp  as  in  the  other  case. 


Cc.  of  N/10 

Cc.  of  N/10 

NaOH  with 

NaOH  with 

Phenol-phthalein. 

Methyl  Orange. 

l6.2 

14.6 

194 

134 

19.8 

13.2 

20.0 

12.5 

20.I 

11.8 

21.2 

11.5 

21.5 

ii-S 

THE  GASTRIC  JUICE  AND  CHANGES  IN  THE  STOMACH.  143 

The  Milk  Curdling  Ferment.  As  has  been  intimated  in  this  and 
earlier  chapters,  two  distinct  views  are  held  concerning  the  coagulation 
of  the  casein  of  milk.  Hammarsten  studied  this  reaction  very  care- 
fully and  ascribed  it  to  the  presence  of  a  peculiar  ferment  which  he 
called  rennin.  It  has  been  explained  that  the  adherents  of  the  Pawlow 
school  consider  this  phenomenon  as  merely  one  of  the  varied  manifes- 
tations of  peptic  digestion,  in  which  casein,  as  the  substratum,  becomes 
first  coagulated  and  then  dissolved  in  part.  The  small  portion  which 
is  left  in  this  peptic  digestion  is  known  as  paranuclein. 

It  has  frequently  been  observed  that  a  coagulum  is  formed  when  a  rennin  solu- 
tion is  added  to  a  crude  proteose  product  from  peptic  digestion.  This  coagulum 
is  called  a  plastein.  But  as  the  precipitate  is  formed  in  other  ways,  as  well,  it  can 
no  longer  be  referred  to  as  a  true  rennin  reaction.  This  plastein  was  at  one  time 
assumed  to  be  a  step  in  the  synthesis  of  larger  groups  from  the  proteoses,  in 
other  words  a  reversed  digestive  process.  How  the  product  is  formed,  or  what  it 
actually  is,  is  not  yet  clearly  known. 

The  Digestion  of  Fats.  It  is  a  discovery  of  comparatively  recent 
date  that  a  lipase  of  considerable  power  is  found  in  the  gastric  secre- 
tion, but  as  to  the  extent  of  the  action  of  this  ferment  in  the  normal 
stomach  but  little  is  yet  known.  The  ordinary  lipase  is  an  intestinal 
enzyme  which  works  in  a  slightly  alkaline  medium,  whereas  this  lipase 
is  said  to  be  active  in  a  weak  acid  medium. 

Absorption  from  the  Stomach.  Among  other  newer  observations 
on  the  functions  of  the  stomach,  it  has  been  shown  that  the  absorption 
of  certain  digestive  products  and  soluble  salts  follows  to  an  appreciable 
extent.  Water  is  not  absorbed  here,  but  under  certain  conditions 
sugars,  peptones,  alcohol  and  bodies  soluble  in  alcohol.  It  has  usually 
been  assumed  that  no  absorption  of  any  importance  is  possible  before 
the  chyme  passes  into  the  intestines,  but  the  later  investigations  on 
dogs  seem  to  show  that  this  view  can  no  longer  be  maintained,  and 
that  the  stomach  may,  in  large  measure,  play  the  part  in  digestion 
which  the  earliest  investigators  ascribed  to  it. 


CHAPTER   IX. 

THE    PRODUCTS    OF    PANCREATIC    DIGESTION. 

After  leaving  the  stomach  where  the  food  is  subjected  to  the  influ- 
ences described  in  the  last  chapter  it  passes  into  the  small  intestine, 
where  it  comes  in  contact  with  other  agents  of  change.  The  work  in 
the  stomach  is  largely  preliminary  and  serves  to  bring  the  food  into  a 
finely  divided  homogeneous  semi-liquid  condition,  in  which  it  may  be 
readily  attacked  by  the  new  digestive  enzymes.  As  explained,  the 
chemical  actions  in  the  stomach  are  comparatively  simple,  and,  leaving 
out  of  consideration  the  continuation  of  the  salivary  digestion,  are  due 
essentially  to  the  combined  effect  of  pepsin,  hydrochloric  acid  and 
protein  substances.  In  the  upper  part  of  the  small  intestine,  however, 
the  work  of  the  pancreatic  enzymes  is  much  more  complicated;  at  least 
three  kinds  of  reactions  take  place  here,  due  to  the  three  distinct  types 
of  ferments  in  the  pancreatic  secretion.  The  protein  digestion  begun 
in  the  stomach  is  completed,  the  carbohydrate  digestion  begun  by  the 
saliva  is  continued  or  completed,  while  the  fats,  not  yet  attacked,  are 
brought  into  a  condition  for  absorption  through  the  intestinal  walls. 
These  three  groups  of  changes  will  be  taken  up  in  detail,  but  something 
must  be  said  first  about  the  pancreatic  juice  as  a  whole. 

COMPOSITION   OF  PANCREATIC  JUICE. 

For  obvious  reasons  it  was  not  possible  to  give  any  fair  analysis  of 
the  gastric  juice.  But  something  more  is  possible  in  the  case  of  the 
pancreatic  secretion  which  may  be  collected  by  means  of  a  fistula. 
Most  of  the  experiments  have  been  made  with  dogs,  and  the  flows,  col- 
lected under  conditions  to  give  a  secretion  as  nearly  normal  as  possible, 
show  that  it  contains  in  the  mean  over  95  per  cent  of  water,  and  solids 
consisting  of  salts  and  organic  substances.  Our  knowledge  of  the 
pancreatic  secretion  has  been  greatly  increased  by  the  work  of  Pawlow, 
who,  by  specially  devised  surgical  methods,  succeeded  in  securing  a 
product  with  little  disturbance  to  the  animal,  and  which  probably  rep- 
resented the  normal  liquid  sufficiently  well.  As  in  the  case  of  the 
stomach  secretion  it  was  clearly  shown  that  the  pancreatic  flow  is 
excited  by  certain  stimuli,  some  of  which  may  be  clearly  followed, 
while  others  are  beyond  explanation,  at  present.  The  entrance  of  the 
acid  chyme  from  the  stomach  into  the  intestines  seems  to  be  the  most 

144 


THE    PRODUCTS    OF    PANCREATIC    DIGESTION.  145 

important  of  these  stimulating  factors;  the  hydrochloric  acid  appar- 
ently aids  in  the  production  of  a  peculiar  ferment  which  enters  the 
blood  and  finally  reaches  the  pancreas.  Other  theories  have  been 
advanced  to  explain  the  manner  of  action  of  the  acid,  but  the  fact  is 
clear  that  the  acid  is  the  most  active  of  all  the  stimulants.  Various 
foods  have  also  marked  effects,  and  the  character  of  the  secretion  fol- 
lowing the  consumption  of  milk,  bread  and  meat  has  been  reported  by 
Pawlow  and  his  pupils.  In  one  set  of  experiments  these  figures  are 
given  for  the  percentage  amounts  of  organic  and  inorganic  solids  in 
the  juice : 

Inorg.  Org. 

Meat  0.907  1.558 

Milk  0.869  4.399 

Bread  0.925  2.298 

In  general,  it  has  been  noticed  that  the  amount  and  nature  of  the 
pancreatic  flow  seem  to  be  adjusted  to  meet  the  requirements  of  the 
peculiar  chyme  furnished  by  the  stomach.  Of  the  mechanism  of  this 
adjustment  practically  nothing  is  known.  The  juice  is  always  alka- 
line, and  the  alkalinity  is  about  that  of  a  sodium  hydroxide  solution  of 
0.5  per  cent  strength.  Of  the  volume  of  the  secretion  in  man  little 
is  accurately  known;  from  fistulas  several  hundred  cubic  centimeters 
daily  have  been  collected,  but  this  may  not  represent  the  normal  flow. 
The  important  enzymes  present  are  trypsin,  lipase  and  amylopsin. 

THE  BEHAVIOR  OF  TRYPSIN. 

In  an  earlier  chapter  a  few  words  were  said  about  the  function  of 
this  important  pancreatic  enzyme  and  it  remains  to  discuss  its  practical 
relations  to  food  digestion.  In  view  of  the  discoveries  of  Pawlow,  it 
seems  probable  that  the  trypsin  is  not  secreted  by  the  pancreas  as  such, 
but  in  the  form  of  trypsinogen,  which  is  activated  by  the  intestinal 
ferment,  to  be  later  described,  known  as  enterokinase.  The  acid 
chyme  from  the  stomach  passing  into  the  intestine  is  neutralized  by  the 
alkaline  pancreatic  fluid  and  the  bile.  In  this  neutralized  condition 
the  trypsin  is  able  to  continue  the  breaking  down  process  begun  by 
the  pepsin,  and  the  proteoses  formed  in  the  stomach  are  carried  further 
to  the  peptone  stage  and  made  ready  for  absorption  or  further  cleavage 
by  erepsin.  From  what  was  said  in  the  last  chapter  it  is  evident  that 
the  trypsin  could  effect  the  preliminary  changes  also;  that  is,  it  is  not 
really  necessary  that  the  food  proteins  should  be  brought  into  the 
proteose  condition  before  the  action  of  trypsin  may  begin.  This  en- 
zyme is  able  to  effect  the  complete  digestion  from  the  beginning,  and 


I46  PHYSIOLOGICAL    CHEMISTRY. 

rather  rapidly  too,  which  may  be  illustrated  by  experiments,  using 
either  the  minced  gland  from  some  animal  or  an  extract  made  by  the 
aid  of  a  proper  solvent.  Such  an  active  extract  may  be  secured  in  sev- 
eral ways.     The  following  methods  answer  very  well. 

DIGESTIVE   EXTRACTS. 

Experiment.  Mince  a  hog's  pancreas  fine  and  weigh  out  about  10  gm.,  which 
cover  with  absolute  alcohol  in  a  small  bottle.  Cork  and  allow  to  stand  over  night. 
Then  pour  off  the  alcohol,  which  is  added  to  remove  water,  and  squeeze  out  the 
residue.  Return  to  the  bottle,  add  10  cc.  of  glycerol  and  allow  the  mixture  to  stand 
about  a  week  with  frequent  shaking.  At  the  end  of  this  time  pour  or  strain  off 
the  glycerol  which  is  now  a  fairly  strong  pancreatic  extract,  and  able  to  act  on  the 
three  classes  of  food  stuffs.  It  has  been  found  by  experience  that  the  extracts 
from  beef  and  hog  glands  are  not  quite  the  same  in  digestive  activity,  but  the  hog's 
pancreas  yields  a  product  suitable  for  all  practical  purposes,  and  which  keeps  a  long 
time  when  made  in  this  manner. 

Experiment.  An  active  pancreas  powder  which  keeps  indefinitely  is  also  very 
useful  and  may  be  made  in  this  way.  Remove  the  adhering  fat  as  carefully  as 
possible  from  a  hog  or  beef  pancreas  and  mince  it  fine  in  a  meat  chopping  mill. 
The  disintegrated  substance  is  treated  as  above  with  an  excess  of  absolute  alcohol 
to  remove  water.  The  alcohol  is  poured  off  and  the  residue  pressed  dry.  This 
residue  is  mixed  with  ether,  allowed  to  stand  an  hour,  and  then  freed  from  ether 
by  pouring,  pressing  and  air  evaporation.  This  treatment  removes  practically  all 
the  water,  traces  of  fat  remaining  and  other  substances  soluble  in  alcohol  and 
ether.  What  is  left  is  thoroughly  air  dried,  ground  to  a  fine  powder  and  sifted 
through  gauze  with  20  to  30  meshes  to  the  inch.  The  powder  so  secured  may  be 
kept  in  a  stoppered  bottle.  In  digestion  experiments  the  powder  may  be  used 
directly,  or  an  extract  may  be  employed.  This  is  best  obtained  by  soaking  a  few 
grams  of  the  powder  with  fifty  times  it's  weight  of  thymol  water  through  24  hours. 

Some  of  the  conditions  of  pancreatic  digestion  may  be  illustrated  by 
very  simple  experiments. 

Experiment.  Pour  25  cc.  of  a  1  per  cent  solution  of  sodium  carbonate  (crys- 
tallized salt)  into  each  of  several  small  flasks  or  test-tubes.  Add  to  each  half 
a  cc.  of  the  glycerol  extract  of  pancreas  and  about  a  gram  of  finely  divided  hard 
boiled  white  of  egg.  (The  white  of  egg  can  be  easily  prepared  according  to  the 
method  given  under  the  pepsin  test.)  Make  one  of  the  tubes  slightly  acid  by  the 
addition  of  dilute  hydrochloric  acid,  enough  to  amount  to  0.2  or  0.3  per  cent.  Now 
place  all  of  them  in  water  kept  at  400  C.  At  the  end  of  half  an  hour  remove  one 
of  the  alkaline  tubes,  and  note  that  it  still  contains  unaltered  coagulated  albumin. 
Test  the  liquid  for  albumoses  and  peptones  as  given  above.  After  another  half 
hour,  test  a  second  tube  (after  filtration).  It  will  be  observed  that  as  the  coagu- 
lated protein  disappears  peptones  become  more  abundant. 

Allow  one  of  the  alkaline  tubes  to  remain  several  hours  at  a  temperature  of  400  C. 
In  time  it  develops  a  disagreeable  odor,  due  to  the  presence  of  indol  formed.  The 
tube  containing  the  hydrochloric  acid  kept  several  hours  at  400  C.  does  not  show  the 
effect  of  digestion,  indicating  that  an  acid  medium  does  not  suffice  for  the  con- 
verting activity  of  the  pancreatic  ferment.  A  minute  trace  of  acid,  below  about 
0.05  per  cent,  does  not  appear  to  check  the  action. 

To  readily  recognize  the  final  products  of  the  pancreatic  digestion  of  proteins 
it  is  necessary  to  start  with  larger  quantities  of  materials  than  are  given  above. 


THE    PRODUCTS    OF    PANCREATIC    DIGESTION.  147 

An  experiment  made  as  above  shows  at  first  digestion  and  finally 
bacterial  putrefaction  as  disclosed  by  the  indol  odor.  A  better  idea  of 
some  of  the  products  formed  in  digestion  may  be  secured  by  operating 
as  follows : 

Experiment.  Mince  50  gm.  of  fresh  fibrin  and  25  gm.  of  pancreas,  mix  and 
cover  the  mixture  with  250  cc.  of  alkaline  thymolized  water,  the  thymol  being  added 
to  check  too  rapid  putrefaction.  Keep  the  mixture  at  40°  two  or  three  days  in  a 
closed  vessel,  the  mass  being  frequently  shaken  or  stirred.  At  the  end  of  the 
digestion  the  alkali  of  the  mixture  is  neutralized  with  a  faint  excess  of  acetic  acid, 
after  which  it  is  boiled  in  a  porcelain  dish  and  filtered.  Some  of  the  fibrin  may 
remain  and  there  will  always  be  some  fat  to  separate  by  the  filtration.  The  filtrate 
is  used  for  the  identification  of  important  products,  some  of  which  are  readily 
recognized,  while  others  are  not. 

PRODUCTS   OF  DIGESTION. 

Of  the  albumose  stage  in  pancreatic  digestion  little  is  known,  as  peptones  seem 
to  be  the  first  recognizable  products.  The  formation  of  peptones  is  greatly  facilitated 
by  the  previous  activity  of  the  stomach  ferments.  The  peptones  of  trypsin  forma- 
tion are  speedily  followed  by  other  products,  the  most  important  of  which  are 
amino  acids.  Different  proteins  break  down  with  very  different  degrees  of  readi- 
ness, some  quickly,  others  very  slowly.  Among  the  important  cleavage  products 
the  following  may  be  referred  to.  Tryptophane  and  tyrosine  seem  to  split  off  in  a 
very  early  stage,  and  may  appear  even  with  the  peptones. 

Tryptophane.  This  name  is  given  to  a  peculiar  product  or  mix- 
ture of  products  found  in  a  pancreatic  digestion  like  the  above.  It  is 
characterized  by  giving  a  marked  violet  red  color  when  mixed  with  a 
little  chlorine  water  or  bromine  water.  The  composition  of  the  trypto- 
phane is  not  yet  known,  but  on  treatment  with  alkalies  at  the  fusion 
temperature  a  mixture  of  several  complex  aromatic  products,  including 
indol  and  pyrrol,  is  obtained.  Quite  recently  the  name  tryptophane  has 
been  given  to  one  of  the  constituents  of  this  mixture  which  has  the 
formula  CnH12N202  and  which  has  been  shown  to  be  indol  amino 
propionic  acid. 

In  a  concentrated  solution  the  addition  of  bromine  or  chlorine  pro- 
duces a  precipitate.  This  may  be  redissolved  only  in  a  very  consid- 
erable excess  of  water.  The  solution  does  not  yield  the  protein  reac- 
tions at  all,  from  which  it  follows  that  the  body  is  an  advanced  decom- 
pose ion  product.    The  substance  is  sometimes  called  protein  chromogen. 

Experiment.  To  recognize  the  chromogen  or  tryptophane  use  two  or  three  cc. 
of  the  above  lilt  rate  from  the  digestion  experiment.  Add  to  the  liquid  some  bro- 
mine water,  drop  by  drop,  shaking  after  each   addition.     Finally  the   desired  color 

appears. 

Tyrosine  and  Leucine.  These  important  amino  acids  have  already 
been  referred  to  when  the  decomposition  products  of  proteins  were 


I48  PHYSIOLOGICAL    CHEMISTRY. 

described.     They  are  formed  abundantly  in  a  prolonged  digestion  like 

the  above  and  may  be  easily  recognized.     Tyrosine  is  paraoxyphenyl- 

a-aminopropionic  acid, 

•OH 

4^CH2CH(NH2)COOH 

and  is  formed  from  most  of  the  protein  bodies  on  digestion.  It  is  not 
formed  in  appreciable  quantity  from  gelatin.  Leucine  is  regarded  as 
a  caproic  acid  derivative,  or  possibly  as  a-aminoisobutylacetic  acid 
(CH3)2CH.CH2.CH(NH2)COOH.  It  is  one  of  the  most  common 
of  the  protein  cleavage  products,  and  is  formed  from  gelatin  also. 
Both  of  these  substances  are  but  slightly  soluble  in  cold  water  and  may 
be  easily  separated  in  crystalline  form. 

Experiment.  To  recognize  the  two  amino  acids  in  the  digestion  mixture  pro- 
ceed as  follows :  Concentrate  the  bulk  of  the  liquid  to  a  volume  of  25  cc.  and  allow 
it  to  stand  in  a  cold  place  several  days.  At  the  end  of  this  time  filter  through 
fine  muslin  or  a  coarse  filter  paper.  The  granular  mass  so  collected  contains  some 
tyrosine  while  the  bulk  of  the  leucine  remains  in  the  filtrate.  Examine  the  residue 
first.  Wash  it  into  a  beaker  with  a  little  cold  water,  allow  to  settle,  decant  and 
wash  again  by  decantation.  Then  add  a  large  volume  of  water  and  enough  am- 
monia to  give  a  marked  odor.  Heat  to  boiling  and  filter  hot.  The  tyrosine  dis- 
solves in  the  alkaline  liquid.  Concentrate  the  filtrate  until  the  odor  of  ammonia 
has  disappeared  and  allow  to  cool;  crystals  of  tyrosine  separate. 

Examine  some  of  these  under  the  microscope.  The  appearance  is  that  of  bunches 
or  sheaves  of  fine  needles.  These  needles  may  be  dissolved  in  alkalies  and  also  in 
hydrochloric  acid  on  the  slide,  which  behavior  distinguishes  them  from  other  some- 
what similar  crystalline  deposits. 

Millon's  Test.  A  very  distinctive  test  is  by  the  use  of  Millon's  reagent,  which 
has  been  already  illustrated.'  Mix  a  little  of  the  crystalline  deposit  with  some  water 
and  Millon's  reagent  in  a  test-tube  and  apply  heat.  A  red  precipitate  forms  after  a 
time  if  much  tyrosine  is  taken.  With  only  a  minute  amount  a  red  color  only  may 
result.  It  will  be  remembered  that  this  reaction  is  not  confined  to  tyrosine  alone, 
but  is  given  by  many  benzene  derivatives  containing  a  hydroxyl  group  attached 
to  the  nucleus.     Hence  phenol  gives  the  test  distinctly. 

By  heating  a  little  of  the  crystalline  residue  with  2  or  3  cc.  of  strong  sulphuric 
acid  solution  follows.  On  adding  a  drop  of  formaldehyde  solution  a  red  color  is 
produced  which  becomes  green  on  heating  further  with  addition  of  some  glacial 
acetic  acid. 

The  solution  left  after  filtering  off  the  tyrosine  is  concentrated  still  more,  which 
finally  causes  a  separation  of  leucine.  The  concentration  is  continued  until  a 
volume  of  about  5  cc.  is  reached.  Crystals  which  have  separated  may  be  examined 
under  the  microscope.  To  the  concentrated  liquid  about  20  cc.  of  alcohol  is  added, 
the  mixture  heated  on  the  water-bath  to  the  boiling  point  and  then  allowed  to  stand 
until  cold.  It  is  then  filtered.  The  filtrate  contains  most  of  the  leucine  present. 
In  the  precipitate  there  is  peptone.  Evaporate  the  alcoholic  liquid  slowly  to  dryness, 
take  up  the  residue  with  water,  add  some  lead  hydroxide  (lead  oxide  with  a  little 
alkali),  boil  and  filter.  From  the  filtrate  remove  the  excess  of  lead  by  means  of 
hydrogen  sulphide,  filter  again  and  concentrate  the  liquid  to  a  small  bulk  for  the 
crystallization  of  the  leucine. 


THE    PRODUCTS    OF    PANCREATIC    DIGESTION.  149 

Examine  some  of  the  leucine  crystals  under  the  microscope.  They  appear  as 
spherical  bunches  of  very  fine  needles.  Often  the  needle  structure  is  not  visible. 
Hydrochloric  acid  and  weak  alkali  solutions  dissolve  the  needles  on  the  slide. 

Leucine  gives  some  marked  chemical  tests.  Dissolve  some  of  the  crystals  in 
water,  add  sufficient  sodium  hydroxide  to  give  a  good  alkaline  reaction  and  then 
a  few  drops  of  copper  sulphate  solution.  The  precipitate  of  copper  hydroxide  which 
forms  at  first  redissolves,  giving  place  to  a  blue  solution  containing  a  compound 
of  leucine  and  copper. 

Leucine  may  be  oxidized  to  yield  valeric  acid.  On  this  behavior  a  test  is  based. 
To  some  of  the  crystalline  residue  containing  leucine  add  3  drops  of  water  and  2 
or  3  grams  of  solid  potassium  hydroxide.  Heat  in  a  test-tube  until  the  alkali 
melts.  The  leucine  decomposes,  giving  off  ammonia.  Allow  the  mass  to  cool,  add 
enough  water  to  dissolve  the  residue  and  then  enough  dilute  sulphuric  acid  to  give 
a  sharp  reaction.  On  applying  heat  the  odor  of  valeric  acid  becomes  evident. 
Through  the  alkaline  oxidation  carbon  dioxide  is  split  off. 

The  Hexone  Bases  and  Other  Bodies.  In  recent  years  much 
attention  has  been  paid  to  the  more  complex  residues  left  on  tryptic 
digestion.  In  this  mixture  the  hexone  bases,  arginine,  lysine  and  his- 
tidine,  are  important  components.  These  are  all  amino  acids  with  six 
carbon  atoms,  and,  because  of  their  constant  occurrence  in  digestive 
mixtures  and  other  products  of  protein  decomposition,  they  must  be 
looked  upon  as  essential  factors  in  the  protein  structure.  Leucine  and 
tyrosine  always  seem  to  accompany  the  hexones  in  these  decompositions. 

Although  by  prolonged  digestion  products  are  reached  which  do 
not  give  the  biuret  reaction,  it  is  shown  by  Fischer  and  others  in  recent 
work  that  residues  remain  which  are  still  relatively  complex.  The 
name  polypeptides  has  been  given  by  Fischer  to  such  residues,  and  their 
relations  to  chemical  substances  of  definite  composition  pointed  out. 
But  even  these  may  be  finally  broken  down  into  simpler  amino  acids. 

Synthesis  of  Polypeptides.  In  the  last  few  years  a  number  of  these  polypeptides 
have  been  produced  by  several  synthetic  processes.  Among  such  bodies  described  by 
Fischer  the  following  may  be  cited  as  illustrations : 

Diglycylglycine,  XH.CH.CO ■  XHCHXO-  NHCH.COOH.  This  is  a  tripeptide 
and,  as  the  formula  shows,  is  formed  by  a  condensation  of  three  groups  of 
aminoacetic  acid. 

Alanylglycylglycine,  CH^HNH^OXHCH^O-NHCH.COOH.  In  this  com- 
pound alanine,  a-aminopropionic  acid,  is  one  of  the  groups  brought  into  the  com- 
bination with  glycine. 

Phewylalanylglycylglycine,  C,H,CILCH\TH2CO-NHCH2CO-NHCH2COOH. 
This  body  is  of  interest  because  of  the  occurrence  of  phenyl  alanine  among  the 
commoner  protein  cleavage  products,  where  reagents  are  used.  Residues  contain- 
ing this  group  appear  t<>  be  much  more  resistant  toward  tryptic  fermentation. 

LeUCYLPBOLINE.     Proline  =  ^-pyrrolidine  carboxylic   acid. 

'  1 1  ;\  yCH,  —  CH-. 

>CIICH.,CHCON< 
CH/  N  If  -CH, 

•Ml,  I 

COOH 


15°  PHYSIOLOGICAL    CHEMISTRY. 

In  this  case  the  synthesis  of  leucine  and  the  pyrrolidine  carboxylic  acid  has  been 
made.  In  trypsin  digestion  residues  containing  the  latter  body  along  with  phenyl- 
alanine s.eem  to  be  characteristic,  especially  where  casein  is  used.  But  these  residues 
are  easily  decomposed  by  hydrochloric  acid  with  separation  of  the  constituent 
amino  acids. 

These  four  artificial  polypeptides  are  among  the  earlier  products  of  laboratory 
synthesis.  In  recent  studies  by  Fischer  and  his  coworkers  the  number  has  been 
greatly  extended. 

Besides  the  hexone  bases  many  simpler  amino  acids  are  always 
found  in  the  digestive  residue;  glutaminic  acid,  aspartic  acid,  alanine, 
amino  valeric  acid,  glycocoll  and  others  have  been  separated.  The 
hexone  bodies  as  end  products  of  definite  composition  are  of  great 
theoretical  importance  because  of  their  relation  to  the  protamines 
referred  to  in  a  former  chapter.  Some  of  these  protamines  break 
down  almost  quantitatively  into  arginine  and  the  other  hexones,  so 
that  the  latter  may  well  be  looked  upon  as  nucleus  structures  which 
unite,  with  loss  of  water,  to  form  the  more  complex  molecules.  These 
diamino  acids  seem  to  bear  about  the  same  relation  to  the  peptones  and 
proteins  that  sugar  bears  to  dextrin  and  starch.  As  in  the  hydrolysis 
of  starch  the  nature  of  the  end  product  depends  on  the  nature  of  the 
agent  of  cleavage,  so  in  proteolysis  the  same  thing  is  true;  acids  and 
enzymes  work  nearly  in  the  same  way,  but  not  absolutely. 

In  this  connection  it  should  be  pointed  out  that  Siegfried  has  separated  by  a  some- 
what peculiar  method  of  treatment  a  number  of  bodies  which  he  calls  trypsin- 
fibrin  peptones  and  pepsin-fibrin  peptones  which  may  be  represented  by  the  following 
formulas : 

trypsin  antipeptone  a   Ci0H17N3O5 

trypsin  antipeptone  £   CnH^NaOs 

pepsin  peptone  a  C21H34N609 

pepsin  peptone  j8  C21H36N6O10 

The  pepsin  peptone  a  seems  to  be  related  to  the  antipeptones  in  this  way : 

C21H34N609  +  H20  =  C10H17N3O5  +  CuH„N,05 

It  is  urged  by  Siegfried  that  the  constant  optical  rotation  of  these  various  products 
is  a  satisfactory  evidence  of  their  constant  composition.  In  connection  with  these 
formulas  the  formulas  of  the  hexone  bodies  may  be  recalled: 

histidine,  C6H9N302 
arginine,  C6H14N402 
lysine,        C6H14N202 

These  compounds  are  relatively  much  simpler  than  the  Siegfried  peptones  and  might 
readily  be  derived  from  them,  with  separation,  at  the  same  time,  of  still  smaller 
molecules. 

The  conception  of  "  end  product "  in  tryptic  digestion  is  evidently  a 
somewhat  indefinite  one.     In  the  last  edition  of  this  book  the  follow- 


THE    PRODUCTS    OF    PANCREATIC    DIGESTION.  151 

ing  sentence  occurs  :  "  Certainly  in  the  animal  body  the  digestive  cleav- 
age cannot  extend  to  the  production  of  these  small  molecules  which 
would  doubtless  be  useless  for  nutrition.  What  is  obtained  in  artificial 
digestions  depends  largely  on  the  time  given  and  the  activity  of  the 
enzyme  employed;  the  term  'end  product'  is  therefore  wholly  relative." 
In  a  few  short  years  our  views  have  been  materially  changed,  and 
largely  through  the  results  of  the  investigations  of  Cohnheim  and 
Abderhalden.  It  has  been  shown  that  these  advanced  cleavage  prod- 
ucts are  sufficient  to  maintain  the  body  in  nitrogen  equilibrium  through 
long  periods,  and  that  they  may  play  a  very  important  part  in  nutri- 
tion.    This  point  will  be  taken  up  again  presently. 

At  one  time  a  great  deal  was  written  about  the  toxicity  of  these 
digestive  products.  A  toxic  effect  was  certainly  observed  on  injection 
of  the  commercial  peptones  into  the  circulation,  but  this  action  seems 
to  be  due  to  the  presence  of  impurities,  and  to  residues  of  the  ferments 
left,  rather  than  to  anything  inherent  in  the  amino  acids  themselves. 
Since  their  behavior  in  nutrition  has  been  shown  the  notion  of  toxicity 
has  been  abandoned. 

Indol  and  Skatol.  In  a  prolonged  pancreatic  digestion,  especially 
in  the  absence  of  the  protecting  thymol  or  chloroform,  these  bodies 
are  always  formed.  Their  appearance  has  nothing  to  do,  however, 
with  the  enzymic  fermentation  which  gives  rise  to  the  other  products. 
They  are  always  products  of  bacterial  decomposition  and  seem  to  be 
produced  by  the  bacteria  from  some  of  the  enzymic  products,  most 
probably  from  tryptophane.     Indol  has  the  composition, 


/CH> 


c.h.C       yen 

Skatol  is  the  methyl  derivative, 


C  ^CH 

nh/ 


Pure  indol  is  a  crystalline  substance  melting  at  520.  Skatol  melts  at 
95  .  Indol  is  oxidized  in  the  body  to  indoxyl,  which  appears  in  part 
in  the  urine  as  indican  or  potassium  indoxyl  sulphate, 

CgH„Nv 

Skatol  suffers  a  similar  change.  More  will  be  said  about  these  reac- 
tions later.     Although  these  bodies  arc  not  true  pancreatic  products,  it 


152  PHYSIOLOGICAL    CHEMISTRY. 

may  be  well  to  illustrate  their  production  in  this  place,  since  they  fre- 
quently appear  in  pancreatic  digestions.     An  experiment  will  show  this. 

Experiment.  Chop  fine  500  grams  of  meat  and  25  grams  of  pancreas  and  allow 
the  mixture  to  stand  exposed  a  day.  Then  mix  with  2  liters  of  water  and  50  cc. 
of  a  saturated  solution  of  sodium  carbonate,  place  in  a  flask  and  keep  at  a  tem- 
perature of  400  through  about  10  days.  Then  transfer  the  whole  mass  to  a  large 
tin  or  copper  can  and  distil  off  most  of  the  liquid.  For  a  complete  separation  500 
cc.  of  water  should  be  added  at  this  stage  and  this  distilled  also.  The  whole  of 
the  distillate  is  now  acidified  with  hydrochloric  acid  and  divided  into  portions  of 
300  cc.  each,  which  are  shaken  out  thoroughly  in  a  separatory  funnel  with  ether. 
For  the  first  300  cc.  of  acid  liquid  about  200  cc.  of  ether  should  be  used.  The 
extracted  aqueous  layer  is  drawn  off  and  a  new  portion  of  300  cc.  added  to  the  same 
ether.  About  50  cc.of  fresh  ether  must  also  be  added.  The  mixture  is  thoroughly 
shaken,  separated  as  before,  and  the  operation  repeated  until  all  the  acidified 
distillate  is  extracted.  The  ether  is  mixed  with  an  equal  volume  of  water  and 
enough  sodium  hydroxide  to  give  a  strong  reaction.  The  alkali  combines  with  and 
holds  the  volatile  acids  which  are  present  while  indol  and  skatol  remain  in  the 
ether  layer.  Separate  as  before,  transfer  the  ether  to  a  flask  and  distil  at  a  low 
temperature.  Drive  off  three-fourths  of  the  ether  and  allow  the  remainder  to 
evaporate  spontaneously.  It  will  not  be  necessary  to  purify  the  residue  in  any 
way.     Dilute  it  largely  with  water  and  apply  the  following  tests : 

Transfer  10  cc.  of  the  dilute  indol  solution  to  a  test-tube  and  add  1  cc.  of  a 
dilute  sodium  nitrite  solution,  mix  thoroughly  by  shaking  and  then  pour  carefully 
a  few  cc.  of  strong  sulphuric  acid  down  the  side  of  the  tube  so  as  to  form  a  layer 
below  the  other  liquid.  At  the  junction  of  the  two  liquids  a  purple  red  color  is 
formed,  which  changes  to  bluish  green  on  neutralization  with  alkali.  This  test 
is  similar  to  the  one  commonly  employed  in  water  analysis  to  detect  the  presence 
of  indol-producing  bacteria.  The  nitrite  solution  used  must  be  very  weak,  prefer- 
ably not  over  0.02  per  cent  in  strength. 

Another  test  is  performed  in  this  way.  A  splinter  of  soft  pine  wood  is  moistened 
with  strong  hydrochloric  acid  and  then  dipped  in  a  weak  aqueous  solution  of  indol. 
The  wood  gradually  becomes  red.  With  much  indol  the  color  becomes  deep  and 
characteristic. 

A  characteristic  test  of  value  depends  on  the  formation  of  a  salt  of  nitroso- 
indol.  Acidify  the  indol  solution  to  be  tested  with  nitric  acid  and  then  add  a  few 
drops  of  a  2  per  cent  solution  of  sodium  nitrite.  The  nitrate  of  nitroso-indol, 
C16H13 ( NO )N2HN03,  forms  and  produces  a  red  precipitate  if  much  indol  is  present. 
If  the  indol  solution  is  weak  a  red  color  only  forms.  By  adding  some  chloroform 
and  shaking,  the  indol  may  be  concentrated  in  the  junction  layer  between  the  two 
liquids. 

By  adding  a  weak  solution  of  sodium  nitroprusside  to  an  indol  solution  a  yellow 
color  is  first  obtained.  The  addition  of  weak  sodium  hydroxide  changes  this  to 
violet,  which,  in  turn,  becomes  blue  by  acidifying  with  acetic  acid.  This  is  known 
as  Legal's  test. 

Skatol  fails  to  give  the  above  tests. 

THE    CARBOHYDRATE   DIGESTION. 

The  pancreas  furnishes  an  enzyme  called  amylopsin  or  pancreatic 
diastase  which  acts  on  starch  or  dextrin  to  form  sugar.  Beginning 
with  starch  we  have  the  gradual  formation  of  maltose  by  hydrolysis. 


THE    PRODUCTS    OF    PANCREATIC    DIGESTION.  1 5  3 

It  has  been  already  pointed  out  that  this  is  not  a  simple  process  but  one 
which  takes  place  in  several  stages,  various  kinds  of  "  dextrins " 
coming  in  between  the  original  starch  and  the  final  sugar.  In  addition 
to  the  enzyme  which  forms  the  malt  sugar  the  pancreas  furnishes,  in 
small  amount,  a  "  maltase  "  which  converts  this  malt  sugar  into  glucose. 
The  action  may  be  very  well  shown  by  means  of  the  glycerol  extracts 
of  pancreas  described  some  pages  back  under  the  head  of  tryptic 
digestion. 

Experiment.  Prepare  a  starch  paste  with  5  gm.  of  starch  to  100  cc.  of  water. 
Mix  10  cc.  of  this  paste,  after  cooling,  with  5  cc.  of  the  pancreatic  extract,  warm 
to  a  temperature  of  35°-40°  C.  and  notice  that  the  paste  soon  becomes  thin  and 
nearly  clear.  After  a  time  test  for  sugar.  Repeat  the  experiment,  using  pancreatic 
extract  which  has  been  boiled  before  mixing  with  the  starch.  The  sugar  reaction 
now  fails  to  appear,  showing  that  high  temperature  destroys  the  activity  of  the 
enzyme,  as  in  the  case  of  saliva.  Note  in  the  solution  of  the  starch  the  dextrin 
stages  which  may  be  followed  by  the  iodine  test.  For  the  complete  conversion  of 
the  amount  of  starch  here  taken  some  hours  may  be  required.  This  depends  on 
the  strength  of  the  pancreas  extract. 

It  is  well  to  vary  the  experiment  by  employing  some  fresh  minced  gland  in  place 
of  the  extract.     The  pancreas  powder  may  also  be  used. 

We  have  then  the  two  principal  reactions  here,  the  formation  of 
malt  sugar  and  the  inversion  of  the  same.  It  is  not  possible  to  isolate 
the  enzyme  which  produces  the  one  reaction  from  that  which  gives 
rise  to  the  other,  but  that  both  are  products  of  the  cells  of  the  pancreas 
has  been  satisfactorily  shown  against  the  view  that  the  inverting 
enzyme  is  furnished  by  the  so-called  intestinal  juice.  It  may  be  recalled 
that  both  reactions  are  hydrolytic. 

It  is  possible  that  the  living  gland  does  not  contain  the  active  fer- 
ment itself,  but  a  pro- ferment  or  zymogen,  which  becomes  active  after 
the  secretion  has  passed  into  the  intestine,  but  a  zymogen  action  has 
not  been  clearly  proven  as  in  the  case  of  trypsin.  In  the  minced  gland 
the  change  appears  to  take  place  through  the  agency  of  air  and  mois- 
ture. There  is  a  marked  difference  in  the  activity  of  the  glands  of 
different  animals,  which  fact  is  practically  recognized  by  the  manu- 
facturers of  the  commercial  products.  The  pancreas  of  the  hog  fur- 
nishes an  enzymic  mixture  richer  in  the  starch  digesting  agents,  while 
the  beef  pancreas  seems  to  be  most  active  in  the  digestion  of  proteins. 
At  the  present  time  some  very  active  "  pancreatic  diastases  "  are  pre- 
pared by  several  firms  in  this  country. 

As  the  proteins  are  prepared  practically  for  final  absorption  from 
the  intestine  by  the  action  of  trypsin,  so  the  remains  of  the  carbohy- 
drates are  brought  into  the  proper  final  condition  by  the  amylopsin 
and  maltase ;  at  any  rate  the  starches  are  so  prepared,  and  maltose  from 


154  PHYSIOLOGICAL    CHEMISTRY. 

any  source  also.  But  as  to  cane  sugar  and  milk  sugar  there  appears 
to  be  some  little  doubt,  several  authors  claiming  that  the  pancreas  does 
not  contain  lactase  or  invertase,  but  that  the  changes  in  these  sub- 
stances, when  not  already  accomplished  by  the  acid  gastric  juice,  take 
place  through  the  agency  of  the  enzymes  of  the  intestine. 

THE  ACTION  OF  THE  PANCREAS  ON  FATS. 

In  the  general  discussion  of  the  subject  of  enzymes  it  was  shown 
that  a  certain  product  of  the  pancreas  called  steapsin  or  lipase  is  active 
in  splitting  neutral  fats  into  glycerol  and  acid.  This  is  a  true  change 
by  hydrolysis  and  in  effect  is  similar  to  the  splitting  by  water  alone 
at  an  elevated  temperature.  In  the  pancreas  the  reaction  may  not  be 
complete,  but  may  extend  only  to  the  separation  of  one-third  of  the 
acid  as  illustrated  by  this  equation  for  stearin : 

rC18H3502  rOH 

C3HB«j  C18H3502  -f-  HOH  =  C3H5-J  C1SH3502  -f-  HC18H3502 
I C18H3502  I  QsH3502 

This  amount  of  liberated  acid  combining  with  the  sodium  carbonate  of 
the  intestinal  juices  produces  a  soap  which  in  turn  aids  in  the  emulsi- 
fication  of  the  rest  of  the  fat  and  thus  prepares  for  its  passage  through 
the  intestinal  walls.  On  the  other  hand,  certain  writers  maintain  that 
the  fat  must  be  essentially  all  split  before  absorption  is  possible.  The 
fatty  acid  and  glycerol  pass  through  the  intestinal  walls  directly  and 
recombine.  The  change  in  this  case  would  follow  through  the  typical 
equation : 

C3H5  (  C1SH3502)  s  +  3H20  =  C3H5  (  OH  ) ,  +  3C18H3602 

The  behavior  of  the  pancreas  may  be  shown  by  experiment,  but  for 
this  purpose  it  is  much  better  to  use  the  fresh  pancreas  than  to  depend 
on  extracts.  The  lipase  seems  to  be  soluble  in  glycerol  to  some  extent, 
but  unless  the  fresh  gland  is  employed  for  the  extraction  the  result 
may  be  unsatisfactory.     These  points  may  be  tested  by  the  student : 

Experiment.  Rub  up  a  part  of  a  pancreas  with  some  fine,  clean  sand  in  a  mor- 
tar to  bring .  it  into  the  condition  of  a  creamy  paste.  Add  some  water,  mix 
thoroughly,  and  use  this  for  the  tests.  Next  melt  some  butter  to  allow  the  curd  and 
salt  to  settle.  Collect  the  clear  butter  fat.  Mix  a  few  grams  of  the  fat  with  an 
equal  volume  of  the  pancreas  paste,  add  some  water  and  a  few  drops  of  chloro- 
form or  toluene  as  preservative.  Keep  the  mixture  at  a  temperature  of  400 
through  a  period  of  several  hours  or  over  night,  and  observe  that  it  gradually 
becomes  acid  through  the  liberation  of  butyric  acid  from  the  butyrin.  This  may 
be  shown  qualitatively  by  means  of  the  reaction  with  rosolic  acid,  a  slightly 
alkaline  solution  with  red  color  changing  to  yellow  on  addition  of  some  of  the 
pancreas-fat  mixture.  It  may  also  be  shown  by  adding  a  few  drops  of  phenol- 
phthalein  to  some  of  the  mixture  and  then  gradually  very  weak  sodium  hydroxide 


THE    PRODUCTS    OF    PANCREATIC    DIGESTION.  155 

until  the  alkaline  reaction  is  secured.  With  a  standard  alkali  solution  the  volume 
used  becomes  a  measure  of  the  amount  of  acid  set  free. 

In  this  form  of  the  experiment  the  butter  fat  is  more  readily  decomposed  than 
are  the  more  solid  neutral  fats.  Indeed  the  lighter  esters,  such  as  ethyl  butyrate, 
are  frequently  used  to  detect  vegetable  lipase  through  the  same  general  reaction. 
If  in  the  experiment  the  butter  fat  used  is  not  neutral  to  begin  with,  it  is  best  to 
add  a  few  drops  of  rosolic  acid  and  then  very  weak  alkali  until  the  color  just 
changes  to  red.     Lipase  is  destroyed  by  heat  as  are  the  other  enzymes. 

Experiment.  The  emulsifying  power  of  the  pancreas  may  be  shown  also.  Grind 
some  fresh  pancreas  to  a  thin  paste  with  a  little  water.  Add  several  grams  of 
this  mixture  to  some  perfectly  neutral  refined  cotton-seed  oil,  about  10  cc,  in  a 
warm  mortar  and  rub  thoroughly  with  a  pestle.  After  a  considerable  time  an 
emulsion  forms  which  will  bear  dilution  with  much  water.  With  common  oil  con- 
taining a  little  free  fatty  acid  the  emulsion  forms  more  rapidly,  but  in  this  case 
the  reaction  may  be  largely  due  to  the  formation  of  soap  first,  from  the  combina- 
tion of  the  fatty  acid  and  alkali  of  the  pancreatic  secretion.  The  experiment 
would  therefore  fail  to  show  the  presence  of  an  enzyme  as  fat  splitter.  For  suc- 
cess here  a  fresh  pancreas  is  necessary. 

A  pure  neutral  fat  suitable  for  such  experiments  may  be  obtained  by  adding 
a  little  caustic  soda  solution  to  some  refined  cotton-seed  oil  and  then  ether.  On 
shaking  thoroughly  the  neutral  fat  dissolves  in  the  ether,  leaving  the  soap  formed 
and  excess  of  alkali  undissolved,  practically.  The  fat-ether  layer  is  poured  off, 
shaken  several  times  with  water  for  the  removal  of  traces  of  soap  or  alkali,  and 
then  slowly  evaporated.     Neutral  fat  is  left  after  the  volatilization  of  the  ether. 

In  these  emulsification  reactions  the  pancreatic  secretion  is  assisted 
by  the  alkaline  bile.  According  to  the  theory  of  the  formation  of 
soaps,  as  a  preliminary  to  absorption  from  the  intestines,  the  bile  must 
act  as  a  very  important  factor,  as  its  alkali  would  be  needed  for  the 
purpose.  The  bile  acids  have  been  shown  to  be  activators  for  the 
steapsin,  and  to  assist  materially  in  the  cleavage  of  the  glycerol  esters. 
In  addition  the  bile  has  a  distinct  solvent  action  on  fatty  acids  which 
may  be  of  help  in  the  ultimate  passage  of  the  fat  products  from  the 
intestine.     The  general  nature  of  the  bile  products  will  be  discussed  later. 

THE  FUNCTION  OF  THE  INTESTINAL  JUICE. 

Closely  related  to  the  action  of  the  pancreatic  diastases  is  the  beha- 
vior of  certain  enzymes  entering  the  small  intestine  from  other  sources, 
especially  from  the  glands  of  Lieberkiihn.  As  these  enzymes  seem  to 
follow  up  and  complete  the  pancreatic  digestion,  they  may  be  briefly 
mentioned  here.  It  should  be  said  first,  however,  that  any  specific 
digestive  action  due  to  ferments  in  the  secretion  of  these  glands  was 
for  a  long  time  denied,  but  there  appears  now  to  be  no  further  question 
as  to  the  actual  behavior  of  the  secretion  in  this  respect. 

Character  of  the  Secretion.  The  flow  into  the  intestine  from  the 
Lieberkiihn  glands  consists  of  a  thin  serum-like  liquid,  holding  in  solu- 
tion protein  bodies  and  salts.     The  reaction  is  strongly  alkaline  because 


I56  PHYSIOLOGICAL    CHEMISTRY. 

of  the  presence  of  sodium  carbonate.  The  amount  of  this  is  sufficient 
to  give  rise  to  an  evolution  of  carbon  dioxide  when  an  acid  is  added 
to  the  secretion  collected  by  a  fistula.  This  alkali  is  doubtless  impor- 
tant in  two  ways;  it  aids  in  the  emulsifkation  of  fats,  and  also  helps 
in  the  neutralization  of  the  remaining  hydrochloric  acid  from  the  gas- 
tric juice  carried  into  the  intestine  with  the  chyme  current. 

The  ferments  appear  to  have  little  or  no  action  on  fats  or  proteins, 
but  work  on  the  residues  of  carbohydrates  only.  Some  chemists  claim 
to  find  in  the  intestinal  juice  a  slight  starch-digesting  power;  others 
deny  that  such  a  behavior  is  possible  and  limit  the  activity  of  the  secre- 
tion to  the  inversion  of  certain  sugars,  especially  cane  sugar  and  malt 
sugar.  Indeed  some  authors  go  so  far  as  to  urge  that  all  of  the  inver- 
sion processes  taking  place  in  the  intestine  are  brought  about  in  this 
way,  while  the  pancreas  can  produce  malt  sugar  only.  Investigations 
of  this  kind  are  attended  with  considerable  difficulty,  which  fact  must 
be  kept  in  mind  when  attempting  to  draw  conclusions  from  apparently 
contradictory  statements,  such  as  are  quoted  above.  All  recent  inves- 
tigations have  shown  this,  that  while  the  intestinal  juice  may  not  be 
the  sole  agent  of  inversion,  it  is  certainly  an  important  agent  in  this 
direction.  The  ferments  present  are  evidently  of  two  types;  one 
resembling  the  invertase  of  cane  sugar  already  described,  while  the 
other  is  of  the  maltase  type. 

The  Secretion  from  Brunner's  Glands.  The  collection  of  the 
product  from  these  small  glands  offers  considerable  technical  difficulty, 
-and  until  recently  no  very  clear  statements  were  found  in  the  literature 
as  to  the  exact  nature  of  the  secretion.  By  taking  special  precautions, 
however,  Glaessner  succeeded  in  securing  the  secretion  free  from  other 
fluids,  and  has  found  that  it  possesses  marked  proteolytic  properties  in 
solutions  of  all  reactions.  The  digestion  of  protein  is  carried  to  the 
stage  where  tryptophane  may  be  easily  recognized.  The  name  pseudo- 
pepsin  may  be  given  to  the  active  enzyme.  A  lipase  and  an  inverting 
enzyme  are  also  present. 

Erepsin.  Comparatively  recently  a  ferment  called  erepsin  has  been 
described  by  Cohnheim  in  the  intestinal  juice.  It  does  not  digest  the 
true  proteins  but  has  the  power  of  splitting  albumoses  and  peptones  as 
far  as  the  mono  and  diamino  acids.  While  trypsin  has  this  power,  it 
is  very  weak  as  compared  with  erepsin,  which  seems  to  be  the  impor- 
tant agent  concerned  in  the  last  change  in  the  proteins.  Under  the  old 
view  of  protein  digestion  there  was  no  place  for  a  ferment  of  this  char- 
acter, as  extensive  cleavage  of  proteins  was  assumed  to  take  place  only 
in  artificial  media.     But  the  newer  views  of  protein  metabolism  and 


THE    PRODUCTS    OF    PANCREATIC    DIGESTION.  157 

protein  synthesis  are  perfectly  consistent  with  the  profound  hydra- 
tion of  the  digesting  material,  which  erepsin  is  able  to  effect.  In  all 
discussions,  then,  of  protein  splitting  in  the  body  erepsin  must  be  con- 
sidered as  one  of  the  most  active  factors. 

Enterokinase.  This  name  has  been  given  to  a  ferment-like  body 
which  occurs  in  the  intestinal  juice  and  which  has  the  power  of  acti- 
vating trypsinogen.  Without  the  presence  of  this  activator,  it  is  held 
by  some  recent  writers,  trypsin  is  not  formed  and  therefore  cannot 
digest  protein.  The  enterokinase  is  not  a  digestive  agent  itself,  but  a 
co-ferment  of  great  importance.  This  ferment  is  one  of  a  class  much 
discussed  recently.  In  many  reactions  two  enzymes  seem  to  be  con- 
cerned, or  perhaps,  better,  an  enzyme  and  an  activator.  These  acti- 
vators are  sometimes  called  kinases,  and  in  some  cases  they  are  not 
actual  enzymes  themselves. 

In  the  brief  discussions  of  the  last  few  chapters  it  has  been  shown 
in  a  general  way  how  the  important  classes  of  food  stuffs  through  the 
action  of  enzymes  in  different  parts  of  the  body  are  gradually  brought 
into  a  condition  suitable  for  assimilation  and  absorption.  They  have 
undergone  digestion  and  are  ready  to  be  carried  through  the  intestinal 
walls  into  the  blood  stream  to  be  used  as  food  for  the  building  up  of 
the  body  or  as  oxidation  material  for  the  production  of  mechanical 
energy  and  heat.  These  various  digestive  processes  differ  in  many 
ways,  but  they  have  this  important  element  in  common  which  must 
be  kept  in  mind :  they  are  essentially  hydrolytic  in  character,  the  addi- 
tion of  water  by  the  enzymes  being  the  essential  feature  in  all  of  them. 
We  have  next  to  consider  some  changes  in  the  intestines  in  which 
hydrolysis  does  not  play  an  important  part. 


CHAPTER    X. 

CHANGES  IN  THE  INTESTINES.    THE  FECES. 

In  an  ideally  normal  condition  of  the  alimentary  canal  after  the  com- 
pletion of  the  digestive  processes  described  in  the  last  chapters,  there 
should  be  practically  nothing  left  finally  in  the  intestines  but  residues 
of  non-nutritive  value,  along  with  broken  down  products  from  the 
digestive  agents  themselves.  Every  trace  of  sugar  or  starch  should 
have  been  brought  into  the  form  of  a  monosaccharide  and  absorbed ; 
every  particle  of  fat  should  have  been  hydrolyzed  or  emulsified  and 
then  carried  into  the  lacteal  circulation ;  while  the  proteins  should  have 
reached  the  form  of  higher  albumoses  or  peptones  and  have  been  like- 
wise absorbed.  The  actual  situation  approaches  this  ideal  condition 
only  approximately.  In  the  first  place  the  foods  we  consume  are  not 
absolutely  pure  fats,  carbohydrates  or  proteins.  They  all  contain  some 
mineral  matter  which  may  escape  the  various  digestive  actions,  and 
they  usually  contain  certain  organic  substances  whicji  are  only  partially 
digestible.  Some  vegetable  foods,  for  example,  contain  relatively 
large  quantities  of  cellulose,  which  is  a  body  related  to  the  carbohy- 
drates but  which  is  not  attacked  by  the  weak  digesting  enzymes.  In 
the  foods  of  animal  origin  there  are  likewise  substances  which  are  very 
difficult  of  digestive  hydrolysis.  This  is  true  of  some  of  the  albumi- 
noids; horn-like  substances,  for  example,  are  practically  not  attacked, 
while  the  cartilaginous  and  similar  bodies  are  but  slowly  changed. 
From  foods  containing  portions  of  such  compounds  a  residue  would 
always  be  left  therefore,  and  in  the  case  of  poor,  cheap  meat  this  residue 
might  be  considerable. 

OTHER    FERMENTATIONS. 

Bacterial  Processes.  But  the  case  is  complicated  by  other  consid- 
erations. Our  foods  carry  hosts  of  acid  and  putrefactive  ferments 
with  them;  and  some  of  these  at  times  work  through  the  stomach  into 
the  intestine,  where  they  start  reactions  of  their  own.  Just  what 
changes  take  place  in  the  small  intestine  depends  on  the  character  of 
the  food.  Following  the  alkaline  zone  where  the  pancreatic  secretion, 
the  bile  and  intestinal  juice  rapidly  effect  the  changes  already  described, 
there  is  a  zone  of  acid  or  neutral  reaction  where  certain  fermentation 
processes  of  bacterial  origin  take  place.  If  the  food  is  rich  in  carbo- 
ns 


CHANGES    IN    THE    INTESTINES.       THE    FECES.  159 

hydrates  this  fermentation  may  be  considerable,  resulting-  in  the  forma- 
tion of  appreciable  quantities  of  lactic,  butyric  and  acetic  acids.  The 
liberation  of  these  acids  at  this  stage  is  a  matter  of  very  considerable 
importance,  since  it  prevents  the  breaking  up  of  not  yet  absorbed  pro- 
tein by  bacterial  putrefaction.  If  the  acids  were  not  present  bacteria 
would  reach  the  small  intestine  in  enormous  numbers  from  the  large 
intestine  and  greatly  modify  the  conditions  there.  While,  along  with 
the  acid-forming  bacteria,  a  few  others  are  always  present  in  the  small 
intestine,  the  real  putrefactive  changes  do  not  begin  to  a  marked  extent 
until  later,  when  what  remains  of  the  food  passes  down  into  the  large 
intestine.  Ordinarily  the  small  intestine  is  devoid  of  disagreeable 
odor,  showing  the  absence  of  putrefactive  changes. 

It  is  evident  therefore  that  the  chemical  nature  of  the  food  is  a 
factor  of  great  importance  in  determining  the  character  of  the  complex 
reactions  which  follow  the  real  digestive  processes  in  the  upper  part 
of  the  intestine.  Here  we  have  normally  the  work  of  enzymes,  and 
this  is  always  followed  by  bacterial  destruction  of  what  is  left.  But 
we  must  distinguish  between  fermentation  changes  and  putrefactive 
changes,  the  former  being  characteristic  of  carbohydrate  food  and  the 
latter  of  protein  food.  As  one  or  the  other  of  these  predominates,  the 
chemical  processes  taking  place  must  vary.  Throughout  the  length  of 
the  small  intestine,  and  in  the  beginning  of  the  large  intestine  active 
absorption  takes  place,  but  between  the  enzymes  and  the  bacteria  a 
struggle  for  the  possession  of  the  field  is  in  progress  all  the  time. 
Theoretically,  without  the  bacteria,  the  foods  would  undergo  complete 
digestion  and  be  practically  all  absorbed,  but  before  this  ideal  condition 
can  be  reached  the  parasitic  bacteria  begin  their  work  and  rob  the  body 
of  part  of  its  food. 

Acid  Fermentation.  Just  when  this  competition  on  the  part  of  the 
acid-producing  bacteria  begins  is  hard  to  say.  Through  the  upper 
third  of  the  small  intestine  the  reactions  are  essentially  those  of  true 
pancreatic  digestion,  and  there  is  at  no  time  a  sharp  line  of  demarka- 
tion  between  this  zone  and  the  following  one.  The  point  in  the  intes- 
tine where  the  acid  fermentations  begin  is  a  fluctuating  one  and  must 
vary  with  the  time  which  has  elapsed  since  the  beginning  of  the  diges- 
tion as  well  as  with  the  character  of  the  food.  The  enzymic  and  acid 
fermentation  zones  must  besides  overlap  each  other;  that  is,  in  the 
central  part  of  the  tract  the  two  kinds  of  changes  must  go  on  simul- 
taneously. Lactic  and  butyric  fermentations  are  favored  by  a  nearly 
neutral  medium,  and  this  is  for  a  time  secured  by  the  slow  neutraliza- 
tion of  portions  of  these  acids  formed  through  the  alkali  of  the  pan- 


l6o  PHYSIOLOGICAL    CHEMISTRY. 

creatic,  the  intestinal  and  the  bile  secretions.  As  the  foods  push  farther 
down  the  neutralizing  action  of  the  alkali  becomes  less  and  less  marked, 
and  finally  the  characteristic  acid  decomposition  becomes  the  principal 
feature. 

In  some  animals  this  acid  fermentation,  to  the  almost  complete 
exclusion  of  putrefactive  changes,  is  easily  recognized.  The  food  of 
the  herbivora  contains  an  excess  of  pentoses,  starches  and  other  carbo- 
hydrates, and  these  produce  sugar  enough  to  furnish  a  large  portion 
for  intestinal  fermentation.  The  feces  of  these  animals  have  not  the 
disagreeable  odors  of  those  of  carnivorous  animals,  where  the  putre- 
factive reactions  are  very  marked  and  the  fermentations  of  very  minor 
importance.  In  animals  with  a  mixed  diet  this  condition  can  be  very 
largely  changed  at  will  by  causing  a  variation  of  the  food  given  them. 

With  the  disappearance  of  the  larger  part  of  the  carbohydrates 
through  absorption  and  acid  fermentation,  the  products  of  fermenta- 
tion being  themselves  partly  absorbed,  the  activity  of  the  putrefactive 
organisms  gains  the  upper  hand  and  large  numbers  of  complex  reac- 
tions follow.  The  nature  of  some  of  the  bodies  produced  in  this  way 
has  been  already  referred  to  and  further  facts  may  now  be  given.  In 
laboratory  experiments  on  pancreatic  digestion  it  will  be  recalled  that 
two  general  results  are  obtainable.  In  working  with  the  pancreas  or 
pancreatic  extract  plus  fibrin  or  casein  we  add  thymol  or  chloroform 
water  if  it  is  desired  to  secure  the  maximum  enzymic  effect,  but  if,  on 
the  contrary,  the  bacterial  as  well  as  the  enzymic  decompositions  are 
desired  this  protective  addition  is  omitted  and  putrefaction  soon  be- 
comes apparent.  In  the  animal  body  the  acid  fermentation  products 
take  the  place  in  a  measure  of  the  chloroform  or  other  substances  used 
in  the  laboratory  experiments.  Indol  and  skatol  have  been  already 
referred  to  as  characteristic  disintegration  products  resulting  from  the 
action  of  bacteria  on  proteins;  there  are  many  others  in  addition  to 
these,  and  most  of  them  are  compounds  of  the  aromatic  group.  Under 
the  conditions  of  their  appearance  in  the  intestines  these  disintegration 
residues  must  be  largely  formed  from  the  albumoses,  peptones,  leucine 
and  tyrosine  of  the  previous  enzymic  digestions ;  in  comparatively  rare 
cases  it  is  possible  that  the  putrefaction  may  take  place  with  portions 
of  left-over  original  proteins  which  for  some  reason  escaped  digestion 
proper.  Phenol,  paracresol,  phenylacetic  acid,  phenylpropionic  acid, 
para-oxyphenylacetic  acid,  glycocoll,  methyl  mercaptan,  hydrogen  sul- 
phide, marsh  gas  and  still  other  substances,  including  various  volatile 
fatty  acids  and  carbon  dioxide,  have  been  found  here  along  with  the 
indol  and  skatol.     These  various  products  are  produced  mainly  in  the 


CHANGES    IN    THE    INTESTINES.       THE    FECES.  l6l 

large  intestine,  and  here  again  we  find  certain  limitations  to  the  extent 
of  the  reactions.  Through  the  small  intestine  the  contents  have  re- 
mained very  soft  and  liquid,  but  in  the  large  intestine  normally  a 
marked  absorption  of  water  takes  place,  from  which  the  contents 
become  thick  and  at  times  almost  hard.  This  loss  of  water  interferes 
greatly  with  the  progress  of  putrefaction.  In  addition  to  this  the  work 
of  the  bacteria  is  hindered  by  the  accumulation  of  the  products  of 
their  own  production.  Some  of  these  products  have  to  a  certain  degree 
a  bactericidal  action  and  tend  to  check  the  more  rapid  bacterial  devel- 
opment. It  follows  therefore  that  when  the  rectum  is  reached  in  the 
downward  progress  of  the  intestinal  contents  there  may  still  be  present 
remains  of  putrescible  matters  which  might  have  been  broken  down  if 
all  the  conditions  had  been  favorable. 

This  brings  us  to  a  consideration  of  the  final  remains  or  the  feces, 
but  first  a  word  must  be  said  about  the  absorption  of  certain  products 
in  the  lower  stretches  of  the  intestine.  Not  only  are  the  normal  diges- 
tive products  taken  up  from  both  intestines,  from  the  small  intestine 
mainly,  but  various  products  of  the  bacterial  reactions  referred  to 
follow  the  same  course.  The  importance  of  this  fact  is  very  great 
from  two  directions  at  least.  The  excessive  production  of  such  a  body 
as  indol  is  always  a  consequence  of  increased  bacterial  activity  which 
is  often  a  pathological  phenomenon.  The  indol  may  escape  partly 
with  the  feces,  but  a  large  portion  is  always  absorbed  and  is  oxidized 
in  the  tissues,  or  in  the  liver  mainly,  from  which  it  passes  into  the 
blood  and  later  into  the  urine,  where  it  is  recognized  in  the  form  of 
indican.  The  amount  of  indican  and  certain  similar  substances  de- 
tected in  the  urine  is  a  measure  then  of  the  extent  of  putrefactive 
changes  going  on  in  the  intestine.  But  it  must  be  remembered  that 
other  putrefactive  processes,  besides  those  of  the  intestine,  may  furnish 
a  small  portion  of  the  indol.  In  this  oxidation  an  atom  of  oxygen  is 
taken  up  and  indoxyl  is  formed : 

C8HTN  +  O  =  C8H0(OH)N. 

This  indoxyl,  like  other  basic  substances,  always  finds  sulphuric  acid 
to  combine  with  to  yield  indoxyl  sulphuric  acid  or  a  salt  of  the  form 


C.H.N- 
K' 


>SO„ 

or  indican. 

Phenol  is  another  product  of  intestinal  putrefaction  and  in  great 
part  passes  also  into  the  circulation  from  the  lower  intestine  to  reach 

12 


l62  PHYSIOLOGICAL    CHEMISTRY. 

the  urine  finally  in  the  form  of  ethereal  sulphate.  Certain  aromatic 
oxy-acids  are  also  formed  in  the  putrefactive  processes,  and  are  likewise 
absorbed.  Other  substances  referred  to  above  as  putrefactive  products 
follow  the  same  course;  in  part  they  escape  with  the  feces,  and  in  part 
they  suffer  absorption  to  be  more  or  less  changed  and  finally  eliminated 
by  the  urine.  The  importance  of  the  two  substances  for  our  purpose 
is  mainly  diagnostic.  They  are  not  absorbed  in  sufficient  quantity  to 
be  poisonous,  but  if  found  easily  in  the  urine  this  points  to  a  more  than 
normal  intestinal  disintegration  of  protein  substances  or  their  deriva- 
tives. If  the  lower  intestine  becomes  for  any  reason  clogged  with 
fecal  products  which  prevent  the  easy  downward  passage  and  escape 
of  the  contents  of  the  small  intestine,  time  is  given  for  the  more  pro- 
longed action  of  the  bacteria,  resulting  in  the  accumulation  of  these 
disintegration  products.  In  nearly  all  conditions  of  high  fever  the 
same  thing  is  observed.  The  urine  test  is  frequently  therefore  a  sug- 
gestion of  an  approaching  pathological  condition,  or  of  an  aggravated 
condition. 

In  another  direction  these  bacterial  products  have  interest  and  im- 
portance. While  the  traces  of  indol,  phenol,  etc.,  found  may  be  quite 
harmless,  it  does  not  necessarily  follow  that  other  things  produced  in 
the  same  way  may  be  equally  harmless.  On  the  contrary,  some  of  the 
putrefactive  products  found  in  the  intestine  are  violent  poisons  and 
their  absorption  constitutes  an  element  of  danger  to  the  body  as  a 
whole.  In  laboratory  experiments  it  is  an  extremely  simple  matter  to 
obtain  from  certain  bacterial  cultures  soluble  products  which  are  very 
toxic.  These  are  the  toxins  formed  by  the  bacteria  and  when  injected 
into  the  circulation  of  animals  are  capable  of  producing  poisonous 
effects.  Similar  bodies  are  undoubtedly  formed  in  the  intestines  if  the 
bacteria  there  present  become  excessive  in  number.  Sometimes  the 
microorganisms  themselves  penetrate  the  intestinal  walls  and  pass  to 
other  parts  of  the  system,  being  collected  finally  by  the  urine.  But  the 
peculiar  poisons  produced  by  them  are  much  more  likely  to  be  absorbed 
into  the  circulation  and  give  rise  to  special  symptoms  at  points  far 
removed  from  the  infected  intestine.  No  one  of  these  intestinal  toxins 
has  been  isolated  and  definitely  recognized,  but  with  them  other  bodies 
are  formed  which  are  readily  detected  in  the  urine,  as  is  the  indican 
referred  to  above.  In  the  urine  of  typhoid  fever,  and  of  other  patho- 
logical conditions  also,  certain  complex  aromatic  products  are  always 
present  which  give  rise  to  the  well-known  reaction  designated  as  the 
diazo  reaction  of  Ehrlich.     When  a  mixture  of  weak  solutions  of 


CHANGES    IN    THE    INTESTINES.       THE    FECES.  1 63 

sodium  nitrite  and  sulphanilic  acid  is  added  to  this  pathological  urine 
under  certain  conditions  it  strikes  a  carmine  to  garnet  red  color,  due 
to  the  formation  of  an  azo  compound  of  some  kind.  The  urine  must 
add  an  aromatic  body,  different  from  those  normally  present,  to  aid  in 
the  formation  of  this  azo  coloring1  substance.  With  normal  urine  an 
orange  color  is  usually  obtained,  but  this  deeper  red  is  characteristic  of 
some  bacterial  product  probably,  of  the  exact  nature  of  which  we  are 
still  in  ignorance. 

THE    FECES. 

Composition.  In  the  lower  intestine  the  absorption  of  water  is 
one  of  the  most  important  of  the  changes  taking  place  and  this  leaves 
what  remains  in  a  semi-solid  condition  ready  for  final  discharge.  The 
amount  of  water  left  in  the  feces  is  quite  variable,  and  although  the 
fecal  mass  may  appear  hard  the  water  content  is  usually  70  to  85  per 
cent.     In  the  thinner  pathological  discharges  it  may  be  much  higher. 

At  first  sight  it  might  appear  that  the  feces  should  consist  mainly  of 
undigested  residues,  and  this  was  long  held  to  be  the  case ;  but  we  now 
know  that  such  substances  may  make  up  the  least  important  part  of  the 
discharge.  The  several  kinds  of  products  present  may  be  roughly 
divided  as  follows : 

1.  Bacteria. 

2.  Products  formed  by  bacteria. 

3.  Remains  of  the  digestive  ferments. 

4.  Epithelium  and  mucus  from  the  intestinal  walls. 

5.  Food  residues  partly  or  wholly  undigested. 

In  the  normal  fecal  discharge  all  these  groups  are  represented,  but 
incidentally  there  may  be  many  other  things  present.  The  bacteria  may 
make  up  one  third  of  the  whole  weight  of  the  feces  at  times.  There 
are  often  substances  which  become  accidentally  mixed  with  the  food 
and  which  are  not  attacked  by  the  digestive  secretions.  There  may  be 
remains  of  various  substances  taken  into  the  body  as  remedies,  for 
example,  oxide  or  sulphide  of  bismuth  from  bismuth  subnitrate,  or 
chalk  or  other  insoluble  substance  taken  in  the  same  way. 

NORMAL    FECES. 

For  comparison  it  is  necessary  to  have  something  as  a  standard,  and 
as  such  a  fecal  discharge  from  a  condition  approaching  starvation 
might  be  taken.  In  such  feces  there  are  no  food  residues,  but  the  other 
things  are  abundantly  represented.  Many  analyses  of  feces  have  been 
made  from  persons  who  for  a  period  of  several  days  had  consumed  no 


164 


PHYSIOLOGICAL    CHEMISTRY. 


food  and  these  give  some  idea  of  the  character  of  the  discharges  which 
might  be  expected  when  the  minimum  of  food  is  consumed  and  no 
more.  It  has  been  calculated  in  this  way  that  about  10  to  12  grams 
daily  is  the  average  normal  discharge  from  a  man  of  70  kilograms 
weight,  due  to  other  sources  than  the  remains  of  food.  Numerous 
attempts  have  been  made  to  find  the  average  composition  of  feces 
from  a  diet  which  contains  just  enough  protein,  fat  and  carbohydrate 
to  keep  the  body  in  normal  condition.  Some  of  the  results  are  given 
in  the  table  below,  in  per  cent. 

Water.  Dry  Subst.  Fat.  Nitrogen. 

Mixed  diet   76.5                23.5  6.2                1.0 

Mixed  diet  85.0                15.0  4.0               0.9 

Milk  diet    71.2                28.8  4.8                1.4 

Milk  diet    77.0                23.0  2.7                0.9 

A  better  idea  of  the  composition  of  normal  feces  from  a  full  and 
varied  diet  is  shown  in  the  following  table,  obtained  from  the  analyses 
of  the  feces  of  six  men  under  observation  in  the  author's  laboratory 
through  a  period  of  four  months.  The  total  feces  were  collected  daily, 
mixed,  in  periods  of  about  a  week  each,  and  analyzed.  The  figures 
below  are  the  means  of  sixteen  analyses,  and  give  a  good  view  of  the 
general  composition.  The  results  are  given  in  grams  for  each  24 
hours,  and  the  so-called  "  crude  fat "  refers  to  the  ether  extract  of  the 
dry  feces,  not  including  the  fat  combined  as  soaps. 


Subject. 


I 
II 

III 

IV 

V 

VI 


Moist 

Dry 

Nitrogen, 

Crude  fat, 

Nitrogen 

Crude  fat 

weight. 

weight. 

per  cent. 

per  cent. 

in  grams. 

in  grams. 

in  moist. 

in  moist. 

2.49 

178 

33-5 

1.4 

2.7 

4.8l 

I40 

37-3 

1-7 

3-9 

2.38 

5-46 

234 

40.9 

I.I7 

2.32 

2.74 

5-43 

112 

25.6 

1.20 

3-04 

1-34 

3-37 

197 

32.1 

I.I7 

1.98 

2.30 

3.90 

157 

33-8 

1.34 

3-25 

2.10 

5-10 

Crude  fat, 
per  cent, 
in  dry. 

144 
14-6 
13.3 
13-2 
12. 1 

I5-I 


The  moist  feces  in  the  adult  may  weigh  from  50  grams  about  to  400 
or  500  grams  daily  in  health,  or  even  more.  The  average  weight  is 
about  150  gm.,  as  illustrated  by  the  above  table.  The  variations  in  the 
values  from  which  these  averages  were  calculated  were  between  70  and 
309  grams  daily.  The  variations  depend  on  the  individual  and  also 
largely  on  the  character  of  the  food.  This  last  is  illustrated  in  the  fol- 
lowing table  from  Konig's  "  Nahrungsmittel,"  where  for  certain  foods 
the  daily  consumption  is  given,  and  also  the  weight  of  the  moist  and 
dry  feces  in  grams. 


CHANGES    IN    THE    INTESTINES.       THE    FECES.  165 

Food.  Feces,  Grams. 

Fresh.  Dry.  Fresh.                    Dry. 

Roast  beef    884  366.8  65.3                  17.7 

Eggs,  boiled   948.1  247.4  42-7                  13.0 

Milk   2438.0  315.0  96.3                 24.8 

Milk   , 4100.1  529.7  174.0                 50.0 

Milk    2291.0  296.0  )  . 

Cheese    200.0  123.8  }  3 

Milk    2209  285.4 


273.7  66.8 

Cheese    517  320.0  )  '  °  ' 

Meat  614  135-9  S 

Bread    450  303.3  V  299.1  46.5 

Bacon     95.6  ) 

Cornmeal    (mush)    750  641.4  108.0  49.3 

Potatoes    3077.6  819.3  635.0  93.8 

Rice    638.0  551.9  194.6  27.2 

Flour  (as  bread)    500  438.8  95.2  23.5 

Carrots    2566  351-6  1092.6  85.1 

Peas    959.8  835.6  927.1  124.0 

It  will  be  noticed  here  that  the  highest  weights  of  feces  correspond 
to  the  high  weights  of  certain  vegetable  foods  which  are  rich  in  cellu- 
lose. Meat  and  milk  in  proper  amount  yield  feces  which  are  not  exces- 
sive, but  with  milk  and  cheese  in  excessive  amounts  the  weight  of  feces 
becomes  large. 

The  mixed  diets  consumed  by  the  subjects  of  the  experiments  from 
the  author's  laboratory  contained  considerable  amounts  of  the  so-called 
breakfast  foods,  as  well  as  fruits  and  vegetables,  and  the  presence  of 
these  always  tends  to  hinder  the  complete  utilization  of  protein.  The 
apparently  high  loss  in  protein  is  not  all  waste,  however,  as  suggested 
above.  It  is  not  possible  to  consume  a  diet  which  is  satisfactory 
through  a  long  period,  and  still  show  no  loss  of  nitrogen. 

In  what  may  be  called  normal  feces  certain  relations  exist  between 
the  nitrogen,  the  fat  and  the  ash,  if  we  understand  by  the  term  "  normal 
feces  "  a  product  containing  no  excess  of  unabsorbed  food,  as  explained 
above.  Such  feces  contain  as  nitrogen  compounds  only  those  sub- 
stances that  are  left  over  from  the  digestive  secretions  or  bacterial  fer- 
ments, or  are  produced  from  the  intestines  themselves,  while  the  "  fats  " 
are  ether-soluble  products  of  similar  origin,  rather  than  the  original 
complex  glycerides.  In  some  cases  recently  reported  the  following 
figures  were  obtained  which  will  serve  as  illustrations.  Three  dogs  of 
similar  character  were  fed  on  a  meat  diet  through  a  number  of  days, 
two  receiving  just  enough  to  keep  them  in  nitrogen  equilibrium,  while 
the  third  received  an  excess  of  meat.  The  results  of  analyses  of  the 
feces  were,  from  the  dried  substance,  in  per  cent  amounts  as  follows: 


1 66  PHYSIOLOGICAL    CHEMISTRY. 


Dog. 

N. 

Fat. 

Ash. 

I 

8.59 

I3.I8 

19.24 

II 

8.8s 

II.46 

22.09 

III 

10.56 

IO.I2 

14.14 

The  high  nitrogen  of  No.  Ill  indicates  an  excess  of  protein;  this 
being  high,  the  fat  and  ash  must  be  correspondingly  low.  The  differ- 
ence in  the  two  kinds  of  feces  becomes  more  apparent  when  it  is 
remembered  that  a  much  higher  factor  must  be  used  in  multiplying 
the  N  values  to  obtain  original  substance  in  III  than  is  probable  for  I 
and  II.  Since  in  III  an  excess  of  protein  is  known  to  be  present  the 
factor  approaches  6.25,  while  for  I  and  II  it  is  probably  not  over  4.5 
or  5.0.  Something  will  be  said  below  about  the  general  character  of 
the  important  fat  and  nitrogen  substances  present  in  the  feces. 

THE  ANALYSIS  OF  FECES  AND  INTERPRETATION  OF  RESULTS. 

This  may  extend  to  the  recognition  of  a  large  number  of  products, 
but  usually  includes  the  detection  and  determination  of  a  few  impor- 
tant ones  only.  Fat  of  some  kind  is  always  present,  hence,  a  quali- 
tative test  is  of  little  value.  The  total  amount  of  fat  must  be  deter- 
mined by  some  kind  of  an  extractive  process.  Nitrogen  is  determined 
generally  by  the  Kjeldahl  method  and  special  tests  are  made  for  pro- 
teins or  their  more  immediate  derivatives.  Occasionally  unchanged 
starch  and  other  carbohydrates  are  present  which  may  be  recognized 
by  methods  given  below.  The  amount  and  character  of  the  ash  is 
sometimes  of  value,  likewise  the  reaction.  Below  a  few  details  will  be 
given  about  some  of  these  tests. 

Separation.  Ordinarily  tests  are  made  on  feces  of  mixed  diets,  but 
frequently  it  is  desirable  to  observe  the  character  of  feces  following 
special  diet  and  not  modified  by  the  product  from  a  previous  or  later 
diet.  To  separate  the  feces  for  such  tests  several  schemes  have  been 
proposed.  It  is  best  to  give  some  inert  substance  at  the  beginning  of 
the  period  which  will  pass  through  the  stomach  and  intestines  un- 
changed, and  at  the  conclusion  of  the  period  of  dieting  the  same  sub- 
stance may  be  given.  Fine  precipitated  or  floated  silica,  powdered 
charcoal,  carmine  and  other  things  have  been  used  in  this  way.  The 
feces  to  be  examined  are  collected  between  the  discharges  of  the  inert 
and  insoluble  limiting  substances.  Various  devices  are  also  used  to 
collect  the  feces  apart  from  the  urine,  which  is  essential  for  exact  tests. 

Reaction.  In  health  the  reaction  of  feces  with  litmus  is  practically 
neutral  in  most  cases,  but  at  times  either  acid  or  alkaline  reaction  may 
be  found.     With  excessive  putrefaction  in  the  lower  intestine  various 


CHANGES    IN    THE   INTESTINES.       THE    FECES.  1 67 

aromatic  products  and  ammoniacal  compounds  are  formed  which  may- 
show  alkaline  behavior  with  indicators  sensitive  in  this  direction.  On 
the  other  hand  free  fatty  acids  may  occasionally  be  present  in  sufficient 
quantity  to  give  a  distinct  acid  reaction.  In  speaking  of  the  reaction 
in  the  intestine  the  conditions  for  the  formation  of  light  fatty  acids 
were  explained.  Putrefaction  on  the  one  hand  and  fermentation  on 
the  other  are  the  important  factors  in  this  connection,  and  these  depend 
in  turn  largely  on  the  diet.  Meat  diet  gives  usually  neutral  or  alkaline 
reaction;  with  excessive  carbohydrates  the  reaction  may  turn  to  acid. 
With  infants  on  mother's  milk  the  reaction  is  commonly  acid,  while 
with  cow's  milk  it  is  neutral  or  alkaline.  These  tests  refer  to  the 
behavior  with  litmus,  but  with  phenol-phthalein,  which  is  not  very  sen- 
sitive with  weak  alkalies,  an  acid  reaction  due  to  carbonic  acid  is  often 
obtained.  This  fact  should  be  kept  in  mind,  since  the  question  of  reac- 
tion is  often  a  question  of  indicators. 

In  testing  for  the  reaction  some  of  the  mixed  feces  is  spread  on  one 
side  of  the  test  paper  by  means  of  a  glass  rod;  the  color  effect  is  looked 
for  on  the  other  side  of  the  paper.  If  the  feces  are  not  quite  moist  it 
will  be  necessary  to  rub  up  with  a  little  water. 

Dry  Residue  or  Solids.  As  explained  above,  the  larger  part  of  the  fecal  dis- 
charge is  always  water.  The  amount  of  solid  matter  is  best  obtained  by  drying 
a  weighed  portion,  at  a  relatively  low  temperature,  in  a  current  of  hydrogen  or  air. 
By  evaporating  over  a  water-bath  there  will  be  always  some  loss  of  volatile  sub- 
stances besides  water.  It  is  very  difficult  to  obtain  a  perfectly  dry  product  on  the 
water-bath  in  most  cases,  especially  if  fat  is  present.  For  most  purposes  it  is  safest 
to  evaporate  a  relatively  large  amount  to  moderate  dryness  on  the  water-bath, 
after  mixing  the  weighed  feces  with  a  little  alcohol.  This  air-dry  product  is 
weighed  and  finely  powdered  and  a  new  portion  is  weighed  out  for  the  final  com- 
plete drying,  at  a  temperature  of  105°  in  the  air  bath.  There  will  be  some  little 
loss  by  volatilization  of  light  acids  and  other  substances. 

For  this  kind  of  work  a  vacuum  drying  apparatus  which  can  be  heated  to  a 
moderate  temperature  renders  good  service.  It  is  also  possible,  where  time  is  not 
an  object,  to  finally  dry  the  air-dried  product  in  a  desiccator  under  sulphuric  acid; 
that  is,  in  the  form  of  drying  apparatus  in  which  the  acid  is  above  and  the  sub- 
stance below.  For  this  purpose  the  air-dried  feces  must  be  thoroughly  powdered, 
or  distributed  in  a  very  thin  layer.  In  some  pathological  stools  there  is  an  abun- 
dance of  fat,  even  to  one-half  of  the  total  solids.  In  such  cases  a  perfect  drying 
is  always  difficult  with  any  process. 

Specific  Gravity.  An  exact  determination  of  this  datum  is  not 
easily  made,  as  the  occluded  gases  interfere  greatly  with  the  test. 
The  normal  specific  gravity  is  about  1.045  to  1-070,  but  may  be  much 
lower  pathologically.  Fatty  stools  may  have  a  specific  gravity  as  low 
as  0.935. 


1 68  PHYSIOLOGICAL    CHEMISTRY. 

THE    TOTAL    FATS. 

In  the  analysis  of  feces  a  number  of  substances  are  included  under 
the  term  "  fat."  In  the  extraction  of  dried  feces  with  some  solvent 
everything  which  goes  into  solution  is  classed  as  crude  fat,  to  be  more 
fully  identified  by  special  tests  later.  Besides  the  fats  proper  feces 
may  contain  fatty  acids  and  their  soaps,  traces  of  lecithin,  cholesterol, 
cholalic  acid  and  other  bodies  soluble  in  ether  or  chloroform.  In  the 
acidified  feces  these  substances  go  into  solution,  the  acids  of  the  soaps 
being  taken  up  also.  In  the  feces  of  adults  the  fatty  acids  combined 
as  soaps  may  make  up  30  to  40  per  cent  of  the  total  "crude  fat," 
obtained  after  acidification. 

For  this  extraction  it  is  customary  to  add  enough  acid  to  impart  a  faint  acid 
reaction  to  the  feces  and  then  evaporate  to  dryness  with  addition  of  sand  or  other 
inert  insoluble  substance.  The  dry  residue  may  be  transferred  to  a  paper  tube  and 
extracted  with  anhydrous  ether  in  the  Soxhlet  apparatus.  A  better  plan  is  to 
spread  a  weighed  portion  of  the  mixed  and  acidified  feces  over  paper  such  as  is 
employed  in  the  well-known  milk  fat  extraction  process.  The  test  is  completed  by 
drying  the  paper  and  extracting  in  the  Soxhlet  tube,  as  in  the  case  of  milk.  The 
results  so  obtained  are  higher  than  those  from  the  ordinary  process  and  the  time 
required  for  extraction  much  shorter.  But  it  is  not  easy  to  obtain  in  this  way 
enough  fat  for  further  study,  as  not  much  more  than  10  gm.  can  be  easily  worked. 

The  amount  of  crude  fat  in  the  dry  feces  is  variable,  but  may  make 
up  in  the  mean  about  25  per  cent  if  the  acids  combined  as  soaps  are 
included.  Much  of  it  under  normal  conditions  must  be  derived  from 
other  sources  than  the  unutilized  original  fat  of  the  foods;  a  portion 
is  always  derived  from  residues  from  some  of  the  intestinal  secretions, 
and  from  organized  elements  thrown  off  from  the  walls  of  the  intes- 
tines. The  extent  of  the  utilization  of  the  food  fat  depends  largely 
on  its  physical  character,  especially  its  melting  point.  The  solid  fats 
with  high  melting  point  are  but  poorly  utilized  as  the  following  figures 
illustrate,  in  which  the  amount  of  loss  in  the  feces  from  different  kinds 
of  fat  is  given,      (v.  Noorden.) 

Melts.  Loss. 

Olive  oil  liquid  2.3  per  cent. 

Goose  fat   250  2.5 

Lard    34  2.5 

Bacon   43  2.6 

Mutton  tallow  49  7.4 

Stearin  plus  almond  oil    55  10.6 

Pure  stearin  (and  palmitin)   60  90.0 

The  free  fatty  acids  do  not  appear  to  be  as  well  absorbed  as  are  the 
neutral  fats,  and  in  general  from  mixed  vegetable  foods  the  fat  loss  in 
the  feces  is  much  greater  than  from  the  animal  foods. 


CHANGES    IN    THE    INTESTINES.       THE    FECES.  1 69 

Pathologically  there  may  be  a  very  great  increase  of  fat  in  the  feces, 
so  great  in  fact  as  to  be  readily  recognizable  by  the  eye.  This  is  espe- 
cially true  in  cases  where  the  flow  of  bile  into  the  intestine  is  diminished 
or  altogether  hindered.  The  fat  in  the  dry  feces  may  then  amount  to 
50  per  cent  of  the  whole.  Any  derangement  of  the  normal  pancreatic 
functions  leads  also  usually  to  an  increase  of  fat  in  the  feces.  In  the 
last  case  protein  and  carbohydrates  would  suffer  also  in  absorption. 

ANALYSIS  OF  THE  CRUDE  FECES  FAT. 

The  method  of  separating  or  extracting  the  crude  fat  has  been 
briefly  referred  to  above.  An  extract  obtained  in  this  way  may  be 
used  for  a  number  of  tests  after  having  been  weighed.  The  recogni- 
tion of  all  the  substances  in  it  is  practically  out  of  the  question,  but 
there  is  no  difficulty  in  making  an  approximate  separation  if  enough 
fat  be  used.  A  simple  heating  test  will  show  the  presence  of  light  and 
volatile  fats,  which,  however,  are  not  usually  present  in  more  than 
traces.  The  following  scheme  will  be  sufficient  for  the  recognition  of 
the  more  important  constituents.  The  extraction  is  completed  in  the 
Soxhlet  apparatus,  the  ether  distilled  off  and  the  residue  dried  and 
weighed. 

Cholesterol.  Cholesterol  is  not  a  fat  chemically  and  therefore  does  not  undergo 
saponification.  This  behavior  makes  it  possible  to  recognize  it.  Add  to  the  crude 
fat  some  alcoholic  potassium  hydroxide,  for  1  gm.  of  fat  about  1.5  gm.  of  the  stick 
alkali  in  25  cc.  of  alcohol.  Boil  under  a  reflux  condenser  half  an  hour  and  then 
drive  off  the  alcohol.  From  the  dry  residue  extract  the  unchanged  cholesterol  by 
use  of  an  excess  of  ether.  The  substance  is  rather  slowly  soluble  and  a  little  soap 
may  be  dissolved  at  the  same  time.  The  result  is  fairly  accurate.  The  ethereal 
solution  of  the  cholesterol  is  evaporated  and  the  residue  weighed.  By  evaporating 
a  little  of  an  ethereal  solution  of  the  substance  on  a  glass  slide  a  residue  is  secured 
which  will  serve  for  microscopic  identification.  Cholesterol  crystallizes  in  large 
thin  plates. 

Cholalic  Acid.  Cholalic  acid  from  the  bile  is  an  important  constituent  of  the 
crude  fat,  provided  this  is  obtained  from  slightly  acidified  feces.  This  acid  may 
be  detected  in  the  soap  left  after  extraction  of  the  cholesterol  as  just  explained. 
The  soap  is  mixed  with  a  little  water  and  acidified  with  dilute  sulphuric  acid  to 
free  all  the  organic  acids.  The  mixture  is  extracted  with  ether  in  a  separatory 
funnel.  After  completed  extraction  the  ether  is  evaporated,  leaving  the  free  fatty 
and  other  acids.  To  these  a  slight  excess  of  barium  hydroxide  solution  is  added 
and  heat  applied  to  form  barium  soaps.  While  warming,  the  mixture  must  be 
well  stirred  or  shaken.  The  separation  to  be  made  depends  on  the  fact  that  the 
barium  soap  of  cholalic  acid  is  soluble  in  about  25  parts  of  hot  water  while  the 
true  fatty  soaps  are  not.  Therefore  on  washing  with  plenty  of  hot  water  the  bile 
acid  soap  dissolves.  By  evaporating  the  solution  to  a  small  volume,  acidifying  with 
dilute  sulphuric  acid  and  shaking  with  ether  the  cholalic  acid  will  pass  again  into 
ethereal  solution,  from  which  it  may  be  recovered  on  evaporation.  The  acid  may  be 
recognized  by  mixing  with  strong  sulphuric  acid.  A  yellow  solution  results  which 
soon  shows  a  green  fluorescence.     The  acid  may  be  identified  also  by  mixing  with 


17°  PHYSIOLOGICAL    CHEMISTRY. 

a  small  amount  of  water  and  a  little  cane  sugar,  and  adding  then  a  few  drops  of 
strong  sulphuric  acid.  A  red  color  develops  which  becomes  purple.  The  sulphuric 
acid  must  be  added  in  just  sufficient  quantity  to  warm  the  mixture  to  about  70° 
or  750  C.     This  test  is  the  Pettenkofer  bile  test. 

Fatty  Acids  Proper.  After  separating  the  cholesterol  and  cholalic  acid  as  just 
described  the  true  fatty  acids  are  left  in  the  form  of  insoluble  barium  soaps.  By 
acidifying  with  a  little  hydrochloric  acid  and  shaking  with  ether  these  acids  go  into 
solution  and  may  be  recovered  by  evaporation  of  the  ether.  The  fatty  acids  of 
any  lecithin  originally  present  are  included,  as  the  lecithin  would  be  decomposed  in 
the  first  saponification.  The  acids  of  soaps  as  well  as  of  neutral  fats  are  also  in- 
cluded if  the  original  extraction  was  made,  as  assumed,  on  acidified  feces. 

Lecithin.  The  separation  of  lecithin  as  such  from  feces  is  not  practicable  but 
the  amount  may  be  estimated  from  the  phosphoric  acid  separated  in  the  saponifica- 
tion. In  the  above  tests  the  glycerophosphoric  acid  would  go  as  barium  salt  along 
with  cholalic  acid  into  the  hot  water  solution.  The  phosphate  could  be  recognized 
or  determined  in  this.  But  it  may  be  estimated  much  more  accurately  by  using 
some  of  the  original  crude  fat.  This  is  mixed  with  some  sodium  carbonate  and 
ashed  carefully.  Then  a  little  saltpeter  is  added  to  complete  oxidation;  the  fused 
mass  is  dissolved  in  water.  The  phosphoric  acid  may  be  determined  by  titration 
with  uranium  nitrate,  or,  better,  with  molybdic  acid  by  the  Pemberton  method.  In 
this  way  it  is  usually  possible  to  secure  enough  phosphate  to  make  an  accurate 
titration,  in  most  cases,  by  starting  with  a  gram  of  crude  fat. 

Soaps.  The  above  tests  give  the  total  acids.  It  may  be  desirable  to  measure 
the  amount  present  in  the  form  of  soaps.  For  this  purpose  a  double  extraction  is 
necessary.  In  one  case  the  feces  are  dried  and  extracted  with  ether  without  pre- 
liminary acid  treatment.  The  soaps,  not  being  ether  soluble,  remain  behind.  Then 
a  second  extraction,  after  acidification,  is  made ;  the  result  gives  the  total  fats 
and  acids  and  the  difference  between  the  two  extractions  shows  the  acid  due  to 
soaps. 

For  those  extractions  the  paper  coil  method  is  very  satisfactory  as  sufficient 
extract  may  be  obtained  from  about  10  gm.  of  moist  feces  for  satisfactory 
weighing. 

CARBOHYDRATES. 

Under  this  term  starch,  sugar,  gums  and  cellulose  must  be  included. 
With  a  vegetable  diet  the  last  named  is  always  present,  while  on  a 
purely  animal  diet  not  one  of  the  group  can  be  found  in  the  feces. 

Starch.  This  substance  is  found  commonly  in  feces,  under  normal 
as  well  as  pathological  conditions,  and  especially  when  the  diet  has  con- 
tained starch  in  the  form  of  coarse  meal,  which  is  difficult  of  digestion. 
This  is  readily  shown  with  cornmeal  and  other  products  containing 
much  cellulose.  On  the  other  hand  with  fine,  well-sifted  flours  in 
which  the  starch  granules  are  easily  turned  into  paste  by  boiling  or 
baking,  the  utilization  of  the  starch  is  usually  much  more  complete. 
For  the  identification  of  starch  several  methods  have  been  applied, 
some  direct,  others  indirect. 

The  recognition  of  starch  by  the  microscope  usually  fails,  as  the  outline  of  the 
granules  is  destroyed  by  the  process  of  cooking.  When  the  cooking  has  been  im- 
perfect, however,  the  granules  may  be  intact  and  in  a  condition  suitable  for 
identification.     The  iodine  test  is  frequently  satisfactory.     For  this  the  feces  are 


CHANGES    IN    THE   INTESTINES.       THE    FECES.  1 7 1 

boiled  with  water  and  filtered.  In  the  filtrate  the  iodine  solution  is  added  as  for 
other  tests.  To  facilitate  the  separation  of  the  starch  from  cell  structures  a  little 
hydrochloric  acid  may  be  added  to  the  boiling  water. 

The  amount  of  starch  is  best  determined  after  conversion  into  sugar.  This  may 
be  accomplished  by  prolonged  heating  with  a  little  hydrochloric  acid,  which  changes 
the  starch  into  glucose.  This  may  be  measured  by  one  of  the  copper  reduction 
processes,  but  not  without  some  difficulty  as  the  solution  is  always  highly  colored. 
The  necessary  precautions  can  not  be  given  here. 

The  normal  starch  content  of  the  feces  is  always  small,  but  in  dis- 
orders of  digestion,  especially  with  diminished  activity  of  the  pancreas, 
more  starch  may  be  found.  In  very  young  children  the  consumption 
of  starchy  foods  is  very  often  followed  by  the  appearance  of  starch 
in  the  feces. 

Sugar.  The  presence  of  traces  of  sugar  in  the  normal  feces  has 
been  frequently  affirmed  and  as  often  denied.  The  larger  part  of  the 
more  recent  evidence  on  the  question  goes  to  show  that  sugar  is  not 
normally  present  even  in  traces.  All  forms  of  sugar  are  very  soluble 
and  easily  absorbed  from  the  intestine.  The  ability  of  sugar  to  escape 
absorption  through  the  whole  length  of  the  intestinal  tract  would  there- 
fore appear  very  problematical.  Even  in  disease  sugar  is  of  rare 
occurrence  in  the  feces  as  far  as  has  been  determined  by  experiment. 
But  the  detection  of  traces  is  not  an  easy  task. 

Pathologically  sugar  seems  to  pass  through  the  intestine  and  escape 
with  the  feces  only  when  the  conditions  for  absorption  through  the 
intestinal  walls  are  reversed.  This  is  the  case  in  diarrhoea  because  of 
the  more  rapid  movement  downward  in  the  intestine  and  because  of  the 
diminished  or  interrupted  flow  from  the  intestine  to  the  blood.  There 
may  be  an  increased  loss  of  proteins  at  the  same  time. 

The  detection  of  traces  of  sugar  calls  for  a  preliminary  extraction,  and  purifica- 
tion of  the  extract  from  substances  which  might  interfere  with  the  copper  or 
analogous  tests.  A  fermentation  test  is  sometimes  made  and  without  preliminary 
treatment.  This  depends  on  the  spontaneous  decomposition  of  the  sugar  by  bacteria 
with  liberation  of  gas  which  is  collected  and  measured.  Starch  present  gives  the 
same  result,  however,  but  not  so  rapidly. 

Cellulose.  This  is  a  common  constituent  of  feces  after  the  con- 
sumption of  vegetable  foods.  Practically  no  digestion  of  cellulose 
takes  place  in  the  small  intestine  of  man,  but  in  the  large  intestine  there 
is  sometimes  a  bacterial  destruction.  The  detection  of  cellulose  is  not 
difficult,  although  the  methods  are  somewhat  complicated.  The  best 
of  the  methods  depend  on  the  solution  of  other  carbohydrates  by  treat- 
ment with  weak  acid  and  then  with  alkali  and  finally  with  water.  The 
mixture  is  filtered  and  I  lie  residue  washed  with  alcohol  and  ether;  it  is 
crude  cellulose  contaminated  with  a  little  ash  and  protein  substance, 


17 2  PHYSIOLOGICAL    CHEMISTRY. 

both  of  which  may  be  determined.     The  appearance  of  cellulose  in  the 
feces  has  of  course  no  pathological  significance. 

Gums.  As  these  are  not  common  articles  of  food  they  do  not  occur 
usually  in  the  feces.  When  they  are  consumed  in  pastry  and  confec- 
tionery they  may  be  found  later  in  the  feces  since  they  are  not  digested 
with  readiness  in  many  cases.  Some  of  the  gums  are  but  slightly 
soluble  and  undergo  pancreatic  digestion  slowly.  Experiments  have 
shown  that  gum  trag'acanth  and  gum  arabic  may  be  found  in  consid- 
erable quantity  after  their  consumption  in  bon-bons. 

NITROGEN   AND    THE   PROTEINS. 

Nitrogen  is  found  in  the  feces  in  many  combinations.  Some  of 
these  represent  residues  from  the  digestive  operations  and  some  are 
found  in  secondary  products  formed  by  bacterial  or  chemical  action. 
Some  of  the  molecular  combinations  are  large,  while  others  are  rela- 
tively small.  No  conclusion,  therefore,  as  to  the  weight  of  the  nitroge- 
nous bodies  can  be  drawn  from  the  nitrogen  found,  but  the  datum  has 
value  from  other  standpoints. 

Total  Nitrogen.  The  total  nitrogen  in  the  feces  may  be  accurately  determined 
by  a  combustion  process,  but  most  readily  by  the  Kjeldahl  process  which  is  now 
everywhere  employed.  This  depends  on  the  conversion  of  the  nitrogen  into 
ammonia  by  prolonged  heating  with  sulphuric  acid  to  which  a  very  little  metallic 
mercury  is  added.  Often  a  mixture  of  pure  sulphuric  acid  and  potassium  sulphate 
is  employed.  At  the  end  of  the  digestion  the  mixture  is  made  alkaline  with  a  slight 
excess  of  ammonia-free  sodium  hydroxide  and  distilled.  The  ammonia  formed  is 
collected  in  standard  acid  and  measured  in  this  by  titration  of  the  excess  of  acid 
with  standard  alkali. 

Even  in  condition  approaching  starvation,  when  no  food  proteins  can 
possibly  be  present,  the  feces  always  show  some  nitrogen,  which,  as 
pointed  out  above,  must  come  from  the  secretions  thrown  into  the 
intestines  and  from  the  remains  of  bacteria  and  their  products.  A  part 
of  this  nitrogen  therefore  has  once  been  absorbed  to  be  later  thrown 
back  into  the  intestine,  which  fact  must  be  kept  in  mind  in  making 
deductions  from  the  nitrogen  found  as  to  the  loss  of  nitrogen  in  assimi- 
lation. Although  usually  overlooked,  the  nitrogen  existing  in  the 
bacterial  cells  is  an  appreciable  quantity  and  often  makes  up  a  good 
fraction  of  the  whole.  It  has  recently  been  shown  that  in  normal  feces 
nearly  one  third  of  the  dry  weight  may  often  be  made  up  of  the  bacteria. 

The  amount  of  nitrogen  excreted  increases  with  the  food  consumed 
in  general,  and  especially  if  this  food  contains  a  large  amount  of  indi- 
gestible substance.  The  nitrogen  of  a  meat  diet  is  always  more  com- 
pletely utilized  than  is  the  nitrogen  of  beans,  for  example,  where  there 


CHANGES    IN    THE    INTESTINES.       THE    FECES.  173 

is  considerable  cellulose  to  disintegrate.  The  nitrogen  in  this  case  is 
largely  in  the  form  of  protein  residues,  and  may  be  detected  as  will  be 
pointed  out  below.  In  pathological  conditions  of  the  digestive  tract 
there  may  be  a  great  increase  of  unutilized  nitrogen.  This  is  more 
especially  true  of  failures  in  the  pancreatic  digestion  than  it  is  of 
failure  in  the  work  of  the  stomach. 

Proteins.  The  most  important  question  to  consider  here  is  that  of 
proteins  themselves  in  the  feces.  Nitrogen  in  other  forms  has  a  far 
different  meaning  since  it  may  represent  bodies  which  have  been 
already  digested  and  absorbed,  and  then  thrown  into  the  intestine  again. 
But  nitrogen  as  protein  represents  practically  waste  in  most  cases. 
Among  the  protein  substances  which  may  be  found  sometimes  in  feces 
these  may  be  mentioned  :  albumins  proper,  casein,  nucleo-proteids,  albu- 
moses,  peptones  and  naturally  more  or  less  of  certain  albuminoid  bodies 
which  are  digested  with  difficulty.  The  certain  detection  of  all  these 
bodies  under  all  conditions  is  not  always  possible  with  our  present 
knowledge.  Some  of  the  simplest  of  the  tests  employed  will  be  briefly 
mentioned.     The  soluble  substances  only  are  considered  here. 

Albumins.  Acidify  the  fresh  feces  with  dilute  acetic  acid  and  extract  with  dis- 
tilled water.  The  acid  prevents  casein  and  mucin  from  going  into  solution  at  the 
same  time.  Filter  through  good  Swedish  paper  and  apply  tests  to  the  filtrate. 
Albumose  and  peptone  go  into  solution  with  this  treatment. 

Albumins  proper  are  coagulated  by  heating  the  filtrate,  while  the  derived  proteins 
do  not  respond  to  this  test.  The  albumins  give  also  the  biuret  test  and  are  pre- 
cipitated by  solution  of  potassium  ferrocyanide  in  presence  of  acetic  acid.  But 
albumose,  not  peptone,  responds  to  the  same  test. 

Albumoses  and  Peptones.  By  extracting  as  above,  coagulating  any  albumin 
possibly  present  and  filtering,  the  filtrate  may  be  used  for  albumose  and  peptone 
tests.  In  the  filtrate  free  from  albumin,  zinc  sulphate  or  ammonium  sulphate  may 
be  used  to  precipitate  albumose.  In  the  filtrate  from  this  peptone  may  be  recognized 
by  the  biuret  test. 

Casein.  This  is  sometimes  found  in  the  feces  of  children  on  a  milk  diet.  To 
recognize  it  these  tests  may  be  made.  The  fresh  feces  may  be  extracted  first  with 
rather  weak  sodium  chloride  solution  to  take  out  soluble  albumins,  then  with  weak 
acid  to  complete  the  removal  of  such  bodies.  The  casein  may  next  be  brought  into 
solution  with  sodium  hydroxide,  not  too  strong,  and  obtained  in  the  filtrate.  In  such 
a  filtrate  acetic  acid  produces  a  voluminous  precipitate  if  casein  is  present,  but 
mucin  is  also  precipitated.  Casein,  however,  redissolves  in  an  excess  of  acetic 
acid  while  mucin  does  not.  After  filtering  the  casein  may  usually  be  thrown  out 
again  by  cautious  addition  of  alkali  to  the  neutral  point,  but  the  precipitate  is  not 
as  characteristic  as  in  the  first  instance. 

Mucin.  This  was  formerly  supposed  to  be  a  common  and  abundant  product 
in  normal  feces,  but  this  is  not  the  case.  Pathologically  mucin  may  be  present  so 
as  to  be  recognizable  by  the  eye.  A  good  chemical  test  for  small  amounts  is  still 
lacking. 

Nucleo-proteid.  By  extracting  normal  feces  with  lime  water  and  acidifying 
with  acetic  acid,  a  bulky  precipitate  is  obtained  usually,  which  was  supposed  to  be 


174  PHYSIOLOGICAL    CHEMISTRY. 

mucin.  It,  however,  contains  phosphorus  and  belongs  to  the  proteid  group.  The 
substance  is  a  normal  product  in  traces  and  can  be  found  in  feces  following  a  diet 
free  from  nucleins.  It  is  therefore  likely  that  traces  of  this  protein  are  brought  into 
the  intestines  from  the  breaking  down  of  the  intestinal  walls.  Pathologically  much 
more  may  be  found,  but  without  having  a  distinct  diagnostic  indication. 

Of  all  the  protein  substances  mentioned,  the  casein,  if  it  occurs  in 
large  quantities  in  infants'  feces,  has  perhaps  the  greatest  importance 
as  pointing  to  imperfect  digestive  power.  There  can  be  no  question, 
of  course,  as  to  its  origin.  Serum  or  egg  albumin  as  such  could  rarely 
be  present  because  such  proteins  are  ordinarily  consumed  in  the  coagu- 
lated condition.  When  the  tests  point  to  the  presence  of  a  true  soluble 
albumin  the  result  shows  probably  the  entrance  of  albumin  from  the 
blood  by  a  reversal  of  the  normal  osmotic  process.  It  must  be  remem- 
bered, however,  that  true  albumin  is  very  rarely  found  in  the  feces. 
Occasionally  a  reaction  due  to  presence  of  pus  or  blood  may  be  obtained, 
but  the  albumose  or  peptone  reactions  are  much  more  frequent.  In 
diarrhoea  stools,  for  example,  where  insufficient  time  is  given  for 
absorption,  these  bodies  may  be  found. 

Insoluble  Proteins.  The  detection  of  coagulated  proteins  and  of 
partly  disintegrated  albuminoids  is  practically  impossible.  Remains 
of  muscle  fibers  or  other  complex  substances  essentially  protein  may 
sometimes  be  recognized  by  the  microscope  but  they  are  beyond  chem- 
ical identification. 

OTHER  NORMAL  AND  ABNORMAL  SUBSTANCES. 

It  will  not  be  necessary  to  discuss  the  occurrence  of  the  various  putre- 
factive bodies  of  bacterial  origin  which  are  always  found  in  the  feces. 
We  have  here  indol,  skatol,  various  phenols  and  aromatic  acids.  Leu- 
cine and  tyrosine  are  occasionally  found,  but  their  presence  is  generally 
pathological,  if  in  quantity  more  than  traces. 

Among  products  of  distinctly  pathological  origin  blood  and  pus  may 
be  mentioned ;  both  yield  albumin  and  the  corpuscles  of  each  may  fre- 
quently be  recognized  by  the  microscope.  It  is  also  possible  to  recog- 
nize the  coloring  matter  of  the  blood  by  the  spectroscope.  It  has  been 
mentioned  that  cholalic  acid,  a  derivative  of  the  two  characteristic  acids 
of  the  bile,  may  be  found  with  the  fats  of  the  feces.  The  bile  acids 
themselves,  glycocholic  and  taurocholic,  are  also  found;  likewise  the 
bile  pigments  or  their  disintegration  products.  Most  of  the  bile  color- 
ing matters  fail  to  be  reabsorbed  from  the  intestine  into  which  they  are 
discharged,  and  must  be  excreted  therefore  by  the  feces,  and  only  in 
small  part  by  the  urine. 


SECTION    III. 

THE  CHEMISTRY  OF  THE  BLOOD,  THE  TISSUES  AND 
SECRETIONS  OF  THE  BODY. 

CHAPTER    XI. 

THE    BLOOD. 

How  Supplied.  The  conversion  of  food-stuffs  into  absorbable 
products  has  been  discussed  in  the  chapters  of  the  last  section.  It 
must  be  shown  now  how  these  products  are  utilized.  Sooner  or  later, 
by  absorption  from  the  stomach  or  the  intestines,  mainly  from  the 
latter  organs,  they  enter  the  blood  stream  through  two  principal  chan- 
nels, the  portal  vein  and  the  lacteal  lymph  vessels  leading  to  the  thoracic 
duct.  Ordinarily  the  amount  of  absorption  from  the  walls  of  the 
stomach  is  not  great ;  only  when  a  very  large  quantity  of  easily  digested 
food  is  present  in  this  organ  or  under  the  influence  of  special  stimuli 
is  the  passage  of  digested  substances  into  the  circulation  here  appre- 
ciable. The  small  intestine  with  its  very  considerable  surface  gives  up 
the  bulk  of  the  absorbable  products  to  the  blood  or  lymph  stream. 

The  digested  fats  pass  essentially  into  the  minute  lymphatic  vessels 
known  as  the  lacteals.  At  the  time  of  digestion  the  contents  of  these 
vessels  consists  of  a  milky  fluid  termed  chyle,  but  at  other  times  the 
lymph  flowing  here  is  nearly  clear.  Minute  capillary  vessels  leading 
to  the  portal  vein  take  up  the  larger  portions  of  the  carbohydrates  and 
protein  bodies  from  the  small  intestine  and  thus  convey  them  to  the 
liver,  where  a  number  of  important  changes  take  place,  the  most  pro- 
nounced being  the  conversion  of  the  sugar  more  or  less  perfectly  into 
glycogen.  These  reactions  will  receive  attention  later.  Beyond  the 
liver  the  hepatic  veins  lead  to  the  general  circulation.  In  this  general 
way  the  nutriments  reach  the  blood  which  is  the  main  channel  of  distri- 
bution, but  this  fluid  is  far  from  being  a  simple  solution  or  mixture 
of  these  nutriments  in  the  condition  in  which  they  leave  the  alimentary 
canal.  The  most  important  of  the  blood  constituents  are,  in  fact, 
chemically  quite  distinct  from  anything  produced  in  the  course  of  diges- 
tion. Certain  organs  of  the  body  have  the  important  function  of 
working  over  these  digestive  products  and  converting  them  into  the 
things  required  in  the  blood.     To  do  this  several  synthetic  reactions 

'75 


I76  PHYSIOLOGICAL    CHEMISTRY. 

are  necessary ;  how  these  are  carried  out  we  do  not  know,  and  in  some 
cases  we  are  ignorant  also  of  where  they  take  place.  In  what  follows 
some  of  the  main  facts  in  this  connection  will  be  given. 

COMPOSITION   OF   THE   BLOOD. 

Quantitative  Variations.  It  is  evident  that  only  an  average  com- 
position can  be  in  general  considered  since  the  fluid  is  in  a  state  of 
constant  change.  Soon  after  a  meal  certain  constituents  would  nat- 
urally be  found  increased,  and  after  a  period  of  fasting  a  deficiency  in 
the  same  would  follow.  From  what  has  been  said  it  is  further  appar- 
ent that  the  blood  of  the  portal  vein  would  be  found  much  richer  in 
some  substances  than  that  of  the  hepatic  vein  or  the  arteries.  It  must 
also  be  remembered  that  the  blood  is  not  a  homogeneous  fluid  but  con- 
sists of  a  true  solution  in  which  are  suspended  certain  cell  structures. 
We  may  therefore  consider  the  average  composition  of  the  blood  as  a 
whole,  or  of  the  corpuscles  on  the  one  hand  and  the  fluid  portion  or 
plasma  on  the  other.  The  specific  gravity  of  normal  blood  varies 
between  1.05  and  1.07;  the  average  specific  gravity  of  the  serum  is 
about  1.03. 

Approximately  the  blood  makes  up  7  to  8  per  cent  of  the  body 
weight;  therefore  in  an  individual  weighing  70  kilograms  the  blood 
weight  would  be  4.9  to  5.6  kilograms.  Of  this  blood  weight  about  60 
per  cent  belongs  to  the  plasma  and  40  per  cent  to  the  corpuscles. 
Among  the  various  recorded  analyses  of  human  blood  as  a  whole  the 
following  may  be  taken  as  best  illustrating  the  mean  composition,  in 
1000  parts. 

BLOOD   ANALYSES. 

Men.  Women. 

Mean  of  1 1  Mean  of  8 

Analyses.  Analyses. 

Water  779  791 

Solids    221  209 

Fibrin   2.2  2.2 

Hemoglobin    134.5  121 .7    • 

Albumin  and  globulin 76.0  76.0 

Cholesterol,  fat,  lecithin   1.6  1.6 

Salts  and  extractives 6.8  7.4 

The  individual  analyses  from  which  these  means  are  taken  show 
rather  wide  variations.  Some  more  recent  analyses  made  in  Bunge's 
laboratory  show  the  distribution  of  the  mineral  matters  and  may  be 
quoted,  the  figures  here  given  referring  to  the  blood  as  a  whole. 


THE    BLOOD.  177 

Man  of  Woman  of 

25  years.  30  years. 

Water 789  824 

Solids    211  176 

Fibrin 3.9  1.9 

Hemoglobin  and  albumins  199-5  164.8 

Salts   7.9  8.6 

The  salt  content  was  made  up  in  each  case  as  follows : 

Man.  Woman. 

Sodium  chloride   2.701  3417 

Sodium  oxide   921  1.862  -\-  potassa 

Sodium  phosphate   457  .267 

Potassium  chloride 2.062  1.623 

Potassium  sulphate   205  .193 

Potassium  phosphate 1.202  .835 


Calcium  phosphate 193 

Magnesium  phosphate    137 


}_ 

7.878  8.615 


These  figures  do  not  show  the  distribution  of  the  salts  between  the 
plasma  and  corpuscles.  In  the  original  analyses  from  which  they  are 
calculated  by  far  the  larger  part  of  the  sodium  salts  was  found  in  the 
plasma,  while  the  potassium  salts  were  found  largely  in  the  corpuscles. 
The  calcium  and  magnesium  salts  occur  mainly  in  the  plasma.  In  the 
blood  the  excess  of  alkali  shown  exists  probably  mainly  as  carbonate. 
All  analyses  seem  to  indicate  a  difference  between  the  blood  of  men  and 
women.  The  male  blood  is  richer  in  solids.  The  female  blood  on  the 
other  hand  appears  to  be  slightly  richer  in  the  mineral  salts. 

Blood  is  characterized  particularly  by  the  peculiar  compound  con- 
taining iron  present,  known  as  hemoglobin.  Many  of  the  tests  for  the 
recognition  or  identification  of  blood  depend  on  this  substance,  which 
is  found  nowhere  else.  As  a  whole  blood  is  distinguished  by  the  phe- 
nomenon of  coagulation  which  is  connected  with  the  fibrin  present. 
Because  of  the  great  importance  of  this  phenomenon  it  will  be  briefly 
discussed  here;  the  details  of  the  subject  belong  to  physiology  rather 
than  to  chemistry  and  are  not  yet  sufficiently  worked  out  for  clear 
elementary  presentation. 

FIBRIN    AND    THE    COAGULATION    OF    BLOOD. 

As  has  been  already  pointed  out  fibrin  is  the  product  resulting  from 
a  certain  reaction  in  which  a  forerunner  or  parent  substance  called 
fibrinogen  is  concerned.  As  it  exists  in  the  blood  vessels  normally  this 
fibrinogen  is  soluble  and  stable,  but  when  the  vessel  is  pierced  and  the 
contents  allowed  to  come  in  contact  with  the  air  the  soluble  fibrinogen 

'3 


I  78  PHYSIOLOGICAL    CHEMISTRY. 

becomes  the  insoluble  fibrin,  which  is  the  well-known  stringy  substance 
described  in  an  earlier  chapter.  A  great  deal  has  been  written  on  the 
subject  of  this  spontaneous  coagulation,  which  is  now  generally  believed 
to  be  brought  about  by  the  action  of  a  peculiar  ferment  formed  by  the 
breaking  down  of  the  white  blood  corpuscles.  From  these  cells  it 
appears  that  a  special  zymogen  which  has  been  called  prothrombin  is 
first  formed;  this  in  the  presence  of  calcium  salts  yields  the  true  fibrin 
ferment,  or  enzyme,  called  thrombin.  It  may  be  easily  shown  that  the 
addition  of  ammonium  oxalate  or  some  other  precipitant  of  calcium 
salts  to  freshly  drawn  blood  will  prevent  its  coagulation.  It  was 
formerly  held  that  the  calcium  compounds  enter  into  a  chemical  com- 
bination as  part  of  the  fibrin  molecule,  but  Hammarsten's  researches 
seem  to  show  clearly  that  the  part  of  the  calcium  is  in  the  formation 
of  the  ferment. 

In  this  coagulation  it  appears  that  a  portion  of  the  original  fibrin- 
ogen is  split  off,  yielding  a  product  known  as  fibrin-globulin,  which 
remains  in  solution ;  that  is,  the  whole  of  the  fibrinogen  does  not  coagu- 
late as  such.  The  coagulation  may  be  prevented  or  greatly  retarded 
by  addition  of  oxalates  as  just  referred  to,  and  also  by  addition  of 
several  other  foreign  substances,  as  acids,  alkalies,  strong  solutions  of 
alkali  salts,  sugar,  gum,  albumose  solutions,  glycerol,  etc.  An  excess 
of  carbon  dioxide  delays  coagulation,  as  shown  by  the  slower  coagula- 
tion of  venous  blood.  Blood  collected  from  a  vein  in  a  polished  vessel 
of  porcelain  or  in  a  vessel  whose  sides  have  been  covered  with  oil  or 
vaseline  coagulates  slowly.  On  the  other  hand  collecting  in  a  vessel 
with  a  rough  surface  hastens  coagulation,  as  does  any  mechanical  agi- 
tation. It  has  been  shown  that  a  polished  platinum  wire  may  be  passed 
through  a  vein  without  inducing  coagulation,  while  a  thread  in  the 
same  position  will  collect  a  layer  of  fibrin. 

The  various  observations  which  have  been  made,  while  not  affording 
a  full  answer  to  the  question  why  the  blood  does  not  coagulate  sponta- 
neously in  the  living  veins  or  arteries,  suggest  several  important  reasons 
to  account  for  this  absence  of  the  reaction.  One  of  the  factors  evi- 
dently present  in  all  ordinary  coagulations  is  contact  with  a  rough 
foreign  substance.  The  foreign  substance  need  not  be  larger  than  the 
specks  of  dust  which  blood  can  gather  from  the  air.  In  leaving  a  vein 
or  artery  blood  naturally  comes  in  contact  with  such  particles,  and  these 
serve  as  nuclei  for  the  beginning  of  coagulation ;  much  as  a  minute  dust 
particle  may  be  sufficient  to  start  crystallization  in  a  strong  solution 
of  alum.  In  the  body  the  blood  is  normally  in  contact  with  vessels 
with  very  smooth  walls.     If  such  a  vessel  be  ligatured  at  two  points 


THE    BLOOD.  I  79 

and  the  sac  thus  formed  be  cut  out  it  will  be  found  that  the  contained 
blood  will  remain  fluid  some  hours  or  days  even.  This  shows  that 
contact  with  living  walls  is  not  the  element  preventing  coagulation. 

Apparently  blood  exists  normally  in  a  very  peculiar  condition  of 
equilibrium,  in  which  not  one  but  several  factors  are  concerned.  The 
same  may  be  said  of  the  equilibrium  of  many  salt  solutions.  Changes 
of  temperature,  the  addition  of  foreign  bodies  in  traces  even,  stirring, 
pouring  from  one  vessel  into  another,  or  contact  with  the  dust  particles 
of  the  air  in  the  one  case  as  in  the  other  may  induce  a  change.  In  the 
living  vessels  of  the  body  as  well  as  after  leaving  the  body  the  equi- 
librium may  be  destroyed  and  a  coagulation  take  place.  This  is  illus- 
trated in  the  intravascular  clotting  after  wounds  in  which  the  vessels 
as  a  whole  may  not  be  impaired ;  injury  to  the  lining  endothelium 
results  in  throwing  foreign  particles  into  the  blood  stream  sufficient  to 
induce  clotting  or  coagulation. 

EXPERIMENTAL    ILLUSTRATIONS. 

Some  of  the  simpler  phenomena  connected  with  the  coagulation  of 
blood  may  be  readily  shown  by  experiment. 

Experiment.  Have  ready  two  test-tubes.  Pour  into  the  first  one  cc.  of  a  cold 
saturated  solution  of  sodium  sulphate,  the  other  is  left  clean  and  dry.  Decapitate 
a  rat  and  allow  two  cc.  of  the  escaping  blood  to  flow  into  the  tube  containing  the 
sodium  sulphate.  The  rest  of  the  blood  is  collected  in  the  dry  tube.  In  a  very  few 
minutes  coagulation  takes  place  in  the  latter  tube,  while  it  is  prevented  by  the 
sodium  sulphate  in  the  former. 

Allow  both  tubes  to  stand  at  rest  a  day  or  two.  In  the  salted  tube  it  will  be 
noticed  that  most  of  the  corpuscles  have  settled  to  the  bottom,  leaving  a  clear  and 
lighter  colored  liquid,  while  in  the  other  tube  the  coagulum  has  begun  to  shrink  into 
a  smaller  mass,  from  which  droplets  of  yellowish  serum  ooze.  The  corpuscles  in 
this  remain  with  the  fibrin. 

Experiment.  Collect  a  quantity  of  slaughter-house  blood  by  running  two  vol- 
umes of  the  latter  into  one  volume  of  saturated  solution  of  sodium  sulphate.  Shake 
the  mixture  and  allow  it  to  stand  at  a  low  temperature  several  days.  Coagulation 
does  not  occur,  but  a  gradual  precipitation  of  the  corpuscles  is  observed,  leaving  a 
yellowish  liquid  known  as  salted  plasma,  which  may  be  poured  off  and  used  for 
various  experiments. 

Experiment.  Pour  a  few  cc.  of  the  salted  plasma  into  a  test-tube  and  dilute  it 
with  several  times  its  volume  of  water.  On  slight  warming  of  the  mixture,  coagu- 
lation follows.  The  effect  of  the  sodium  sulphate  is  to  prevent  coagulation.  In  this 
case  dilution  favors  it. 

Experiment.  Pour  some  fresh  blood  into  a  clean  vessel  and  stir  it  thoroughly 
with  a  glass  rod,  if  a  small  quantity  in  a  beaker  is  taken,  or  with  a  stick  if  a 
larger  volume,  as  of  slaughter-house  blood,  is  used.  The  fibrin  gradually  separates, 
and  entangles  most  of  the  corpuscles.  Save  the  serum  for  tests  to  be  explained  and 
wash  the  crude  fibrin  thoroughly  under  running  water  to  remove  the  corpuscles  and 
coloring    matter.     The    well-washed    fibrin    is    white    and    stringy.     Fibrin    so    pre- 


l8o  PHYSIOLOGICAL    CHEMISTRY. 

pared  is  employed  in  many  experiments,  especially  in  illustrating  digestion  phe- 
nomena.    On  the  large  scale  it  is  used  in  the  manufacture  of  peptone. 

Experiment.  To  illustrate  the  ready  digestion  of  fresh  fibrin  use  about  half  a 
gram  with  10  cc.  of  0.25  per  cent  hydrochloric  acid.  Keep  the  mixture  some  hours 
at  40°  C.  The  fibrin  gradually  dissolves  to  form  acid  albumin,  which  may  be  ob- 
tained in  solution  by  filtering  from  any  undigested  residue.  The  careful  addition 
of  a  little  sodium  carbonate  solution  produces  a  precipitation  of  the  acid  albumin. 

Time  of  Coagulation.  It  has  long  been  observed  clinically  that  the  time  required 
for  the  coagulation  of  a  drop  of  blood  withdrawn  by  a  needle  is  not  constant  but 
varies,  and  in  a  marked  manner  in  certain  diseases.  Based  on  this  observation 
several  forms  of  apparatus  have  been  devised  in  which  the  rapidity  of  coagulation 
may  be  followed  and  measured.  One  of  the  best  known  forms  it  that  of  Wright, 
which  consists  of  a  number  of  small  glass  tubes  of  uniform  bore,  and  open  at  both 
ends,  into  which  definite  volumes  of  the  blood  in  question  may  be  drawn.  After 
being  filled  with  blood  the  tubes  are  immersed  in  warm  water  of  body  temperature, 
or  in  some  cases  at  a  lower  definite  temperature.  From  time  to  time  a  tube  is 
removed  from  the  bath  and  tested  by  blowing.  As  soon  as  coagulation  begins  the 
blood  can  no  longer  be  blown  out  easily,  and  the  time  required  for  this  is  noted. 
In  health  this  time  may  be  four  or  five  minutes  usually,  but  in  jaundice  and  some 
other  diseases  it  may  be  much  longer.  The  time  varies  somewhat  with  the  form  of 
apparatus  used.  In  the  Boggs  coagulometer  the  time  required  for  the  clotting  of  a 
drop  of  blood  of  definite  size  and  shape  is  followed  under  the  microscope. 

BLOOD    TESTS. 

The  serum  left  after  separation  of  the  fibrin  by  stirring,  contains 
much  of  the  blood  coloring  matter  and  may  be  used  as  well  as  the  fresh 
blood  for  many  tests,  some  of  which  will  be  illustrated  here. 

Experiment.  Guaiacum  Test.  To  a  little  blood  solution  in  a  test-tube  add  some 
fresh  tincture  of  guaiacum  and  then  a  few  drops  of  an  ethereal  solution  of  hydrogen 
peroxide.  Shake  the  mixture  and  observe  that  the  precipitated  resin  has  assumed  a 
blue  color,  more  or  less  marked.  In  this  test  turpentine  oil,  which  has  been  shaken 
with  air  in  a  bottle,  or  which  has  been  exposed  to  the  air,  can  be  used  instead  of 
the  solution  of  peroxide.  Hydrogen  peroxide  is  developed  by  the  action  of  oxygen 
on  turpentine.  In  this  test  the  hemoglobin  seems  to  act  as  a  carrier  of  oxygen  to 
the  resin.     The  oxidation  product  of  the  resin  is  blue. 

Experiment.  Hydrogen  Peroxide  Test.  A  reaction  somewhat  similar  to  the 
above  in  principle  is  observed  on  mixing  2  cc.  of  the  blood  with  10  cc.  of  the  com- 
mercial hydrogen  peroxide  solution.  The  hemoglobin  brings  about  the  decomposition 
of  the  peroxide  with  liberation  of  oxygen,  which  escapes,  producing  froth. 

Reaction  of  Blood.  The  normal  reaction  of  blood  is  alkaline, 
which  cannot  be  observed,  however,  in  the  usual  way  because  of  the 
marked  color  of  the  pigment.  It  may  be  readily  seen  by  working  in 
the  following  manner : 

Experiment.  Prepare  some  small  plaster  of  Paris  surfaces  by  pouring  the 
well-known  plastic  mixture  of  plaster  of  Paris  and  water  on  glass  plates  and  allow 
it  to  harden  several  hours  at  least.  The  prepared  plates  are  removed  from  the 
glass  and  soaked  in  a  neutral  solution  of  litmus  and  are  then  allowed  to  dry.  The 
test  proper  can  now  be  made  by  putting  a  few  drops  of  the  blood  on  the  smooth 
plaster  surface  and  allowing  it  to  remain  there  five  minutes.     It  is  then  washed 


THE    BLOOD. 


181 


off  with  pure  water,  when  it  will  be  found  that  the  part  of  the  plate  which  has  been 
covered  by  the  blood  has  become  blue  from  the  action  of  the  alkali  of  the  blood  on 
the  neutral  litmus. 

Experiment.  Heat  Test.  Heat  the  solution  of  blood  until  it  is  near  the  boiling 
temperature  and  note  that  the  red  color  is  largely  destroyed  and  that  a  brownish 
precipitate  forms  which  contains  albumin  and  decomposed  coloring  matter.  Add 
now  a  small  amount  of  sodium  hydroxide  solution  and  observe  that  the  precipitate 
disappears  while  the  blood  solution  becomes  red  again  by  reflected  light,  but  greenish 
by  transmitted  light. 

Experiment.  Hemin  Crystals.  When  acted  on  by  acids  or  strong  alkalies 
hemoglobin  of  blood  is  broken  up  into  globin  and  a  characteristic  compound 
called  hematin.  Hematin  in  turn  is  acted  upon  by  hydrochloric  acid  yielding 
the  hydrochloride,  hemin,  which  appears  in  crystalline  form.  From  the  name 
of  their  discoverer,  these  crystals  are  called  "  Teichmann's  crystals."  Their  appear- 
ance constitutes  one  of  the  best  tests  we  have  for  blood,  and  can  be  illustrated  by 
the  following:  Evaporate  a  drop  of  blood  on  a  slide,  add  two  or  three  drops  of 
glacial  acetic  acid,  and  boil.  Put  on  a  cover  glass  and  allow  to  cool.  Minute 
(microscopic)  plates  or  prisms  separate  out.  If  old  blood,  a  stain,  for  instance,  is 
examined,  it  is  necessary  to  add  a  small  crystal  of  sodium  chloride  to  the  acetic 
acid,  by  which  means  sufficient  hydrochloric  acid  is  liberated  for  the  test.  The 
crystals  have  a  dark  brown  color  and  are  very  characteristic.  The  usual  forms  as 
found  in  human  and  other  blood  are  shown  below. 


Fig.  q.       Hemin   crystals.       i    is   from   human 

blood ;  2  from  a  seal ;  3  from  a  calf ;  4  from  a 

pig;   5  from  a  lamb;   6  from  a  pike;   7  from  a 

rabbit.  (Landois.) 


x'K-»' 


v»    V.v 


V 

\     » 


\ 


Fig.  10.  Hemin  crys- 
tals from  stains  of  hu- 
man blood.      (Landois.) 


The  most  certain  means  of  identifying  blood,  however,  depends  on 
the  peculiar  behavior  of  hemoglobin  toward  light,  which  will  be  shortly- 
explained. 

HEMOGLOBIN. 

Composition.  In  the  systematic  classification  of  the  protein  bodies 
hemoglobin  is  grouped  among  the  proteids  or  compound  substances, 
inasmuch  as  it  may  readily  be  broken  up  into  a  fraction  containing  iron 
called  hematin,  and  a  histone  substance  called  globin.  This  cleavage 
is  very  easily  effected  by  the  action  of  weak  acids  and  in  the  mean  the 
hematin  fraction  is  found  to  amount  to  about  4.3  per  cent.     In  some 


182 


PHYSIOLOGICAL    CHEMISTRY. 


experiments  as  much  as  94  per  cent  of  globin  has  been  recovered.  It 
is  therefore  likely  that  only  the  two  substances  are  present.  The  prop- 
erties of  hemoglobin  are  not  quite  constant,  inasmuch  as  from  different 
bloods  products  of  slightly  different  composition  have  been  obtained. 
It  is  possible  to  secure  the  hemoglobin  in  crystalline  condition  suitable 
for  analysis.  A  number  of  such  determinations  have  been  made  and 
from  them  formulas  have  been  calculated.  These  formulas  can  be  at 
best  only  more  or  less  close  approximations,  but  they  are  interesting  as 
illustrating  the  great  molecular  weights  here  concerned. 

Hemoglobin  is  dextro-rotatory.  By  an  ingenious  method  Gamgee 
and  Hill  have  found  ac=  10.40.     The  globin  from  it  is  levorotatory. 

Analyses  of  Hemoglobin.  Several  results  obtained  by  different 
observers  are  here  given.  The  variations  must  be  partly  due  to  differ- 
ences in  methods  of  preparation  and  analysis. 


C 

H 

N 

s 

Fe 

0 

Author. 

Horse. 

51-15 

6.76 

17-94 

0.39 

o-335 

23.42 

Zinnofsky. 

" 

54-40 

7.20 

17.61 

O.65 

0.47 

19.67 

Huefner. 

Dog. 

54-57 

7.22 

16.38 

0.57 

Q-33D 

20.43 

Jaquet. 

(  ( 

53-85 

7-32 

16.17 

0.39 

o.43 

21.84 

Hoppe-Seyler. 

Hen. 

52.47 

7.19 

16.45 

O.86 

o-335 

22.5 

Jaquet. 

In  the  first  analysis  the  ratio  of  the  sulphur  atoms  to  the  iron  atoms 
is  2:1;  in  the  third  analysis  it  is  3:1.  On  the  assumption  that  the 
molecule  contains  but  one  atom  of  iron  the  minimum  molecular  weight 
which  may  be  calculated  from  this  analysis  is : 

^758-H-:i203-N  las'-^is-t1  e^>3 

It  is  interesting  to  note  that  the  molecular  weights  found  in  this  way 
are  practically  confirmed  by  the  determinations  made  on  the  combining 
power  of  hemoglobin  for  carbon  monoxide.  Assuming  that  one  mole- 
cule of  carbon  monoxide  is  held  by  one  molecule  of  hemoglobin,  obser- 
vations of  the  volume  of  the  gas  absorbed  by  a  given  weight  of  the 
blood  pigment  lead  to  practically  the  same  result  as  was  obtained  by 
the  iron  method. 

Combinations  of  Hemoglobin.  The  great  importance  of  hemo- 
globin depends  on  its  power  of  forming  several  more  or  less  stable 
combinations  with  certain  gases.  Of  these  combinations  that  with 
oxygen  is  by  far  the  most  important;  we  distinguish  therefore  between 
hemoglobin  and  oxyhemoglobin.  The  common  form  of  the  substance 
is  really  the  latter,  although  it  is  usually  referred  to  by  the  simple 
term — hemoglobin.  The  oxygen  of  oxyhemoglobin  is  very  loosely 
held  and  may  be  driven  out  from  its  union  by  the  aid  of  a  current  of 


THE    BLOOD. 


I83 


other  gases,  or  by  the  pump.  The  amount  so  held  corresponds  to  two 
atoms  of  oxygen  for  each  molecule  of  hemoglobin.  This  oxygen  com- 
bining power  in  some  way  depends  on  the  presence  of  the  iron  of  the 
hematin. 

Oxyhemoglobin.  By  various  methods  this  substance  may  be  ob- 
tained in  crystalline  form,  the  crystals  being  often  2  mm.  or  more  in 
length.  From  blood  of  different  animals  different  crystalline  forms 
have  been  observed.  In  all  cases  the  crystals  are  red  and  soluble  in 
water ;  they  are  more  easily  soluble  in  water  containing  a  little  sodium 
carbonate.  They  are  insoluble  in  ether,  benzene  and  chloroform,  and 
the  water  solubility  varies  with  the  nature  of  the  blood  from  which 
they  were  made,  that  from  the  blood  of  man  and  the  ox,  for  exam- 
ple, being  easily  soluble,  while  the  oxyhemoglobin  from  the  blood  of 
the  horse  or  dog  is  rather  slowly  soluble.  Because  of  their  solubility 
it  is  very  difficult  to  secure  crystals  from  human  blood,  but  from  dog's 
blood  they  may  be  made  as  follows : 

Experiment.  Beat  up  100  cc.  of  the 
blood  thoroughly,  cool  to  a  low  tem- 
perature and  add  10  cc.  of  ether  and  a 
little  water.  Shake  this  mixture  thor- 
oughly and  allow  it  to  stand  on  ice  over 
night.  Filter  on  porous  paper,  squeeze 
out  the  mother-liquor  as  far  as  possible, 
dissolve  in  a  little  water,  filter  again 
and  to  the  new  filtrate  add  one-fourth 
its  volume  of  alcohol,  meanwhile  stirring 
constantly.  Allow  the  mixture  to  stand 
to  crystallize.  An  illustration  is  given 
of  the  usual  forms. 

A  simple  test  may  be  also  made  by 
mixing  a  drop  of  dog's  blood  with  a 
drop  of  water  on  a  slide  and  allowing  it 
to  partly  evaporate.  A  cover  glass  is 
then  put  on  and  the  crystals  are  looked 
for   with   the  microscope. 

The  conditions  of  combination 
between  hemoglobin  and  oxygen 
have  been  studied  by  several  au- 
thors. It  has  been  found  that  by 
exhausting  blood  under  the  air 
pump  the  greater  part  of  the  oxy- 
gen becomes  free.  It  has  been 
found  further  that  1  gm.  of  hem- 
oglobin may  be  made  to  take  up  or  give  off  something  over  1.3  cc.  of 
'^en.     This  reduces  to  2  atoms  of  oxygen  for  1  atom  of  iron  pres- 


Fig.  II.  Crystals  of  hemoglobin,  a  and 
b  from  human  blood ;  c  from  the  cat ;  d 
from  the  guinea-pig ;  e  from  the  marmot ; 
/  from  the  squirrel. 


1 84  PHYSIOLOGICAL    CHEMISTRY. 

ent  in  the  hemoglobin.  Various  chemical  agents  have  the  same  effect. 
In  the  case  of  certain  solutions  the  action  is  a  chemical  one,  while 
with  several  inert  gases  the  action  is  physical. 

These  reactions  are  accompanied  by  a  change  of  color  in  the  oxyhe- 
moglobin or  blood  solution  experimented  upon.  Oxyhemoglobin  solu- 
tions show  a  brighter  red  color  than  do  those  containing  the  reduced 
hemoglobin.  This  difference  is  well  illustrated  in  the  contrasting 
shades  of  arterial  and  venous  blood,  the  former  containing  plenty  of 
oxygen  in  combination  while  the  latter  is  deficient.  The  loss  of  oxygen 
is  illustrated  by  some  simple  experiments : 

Experiment.  Shake  about  10  cc.  of  diluted  defibrinated  blood  with  a  few  drops 
of  ammonium  sulphide  solution  or  with  Stokes'  reagent.  (Stokes'  reagent  is  a 
solution  of  ferrous  sulphate,  to  which  a  small  amount  of  tartaric  acid  has  been 
added,  and  then  ammonia  enough  to  give  an  alkaline  reaction.)  Warm  gently,  and 
observe  that  the  bright  color  of  arterial  blood  gives  place  to  the  darker  purple  of 
venous.  On  shaking  the  mixture  now  with  air  the  bright  red  color  returns.  For  the 
success  of  this  experiment  where  Stokes'  reagent  is  employed  it  should  be  freshly 
prepared  before  use.     Various  other  substances  behave  in  similar  manner. 

Experiment.  Generate  some  hydrogen  gas  in  the  usual  manner,  and  allow  it  to 
bubble  through  diluted  defibrinated  blood.  A  change  of  color  follows  after  a  time, 
due  to  the  mechanical  loss  of  oxygen.  The  same  .result  may  be  accomplished  by 
exhausting  the  oxygen  of  the  blood  by  means  of  an  air  pump.  Exposure  to  the 
air  restores  the  color  in  a  short  time,  as  before. 

The  color  change  may  be  noticed  readily  with  the  unaided  eye,  but 
is  much  more  marked  when  observed  in  the  spectroscope,  as  will  be 
pointed  out  below. 

The  maximum  amount  of  oxygen  which  may  be  held  by  the  hemo- 
globin was  given  above  as  i  molecule  for  I  molecule  of  the  pigment. 
This  holds  only  for  strong  oxygen  pressure,  however.  Under  lower 
atmospheric  pressure  a  part  of  the  oxygen  becomes  dissociated,  as 
illustrated  by  these  figures  given  by  Huefner  for  a  14  per  cent  hemo- 
globin solution : 


Atmospheric 

Per  cent  of 

Atmospheric 

Per  cent  of 

Pressure  in  Mm. 

Oxygen  Free. 

Pressure  in  Mm. 

Oxygen  Free. 

760.O  mm. 

1.49 

238.5  mm. 

4.60 

715-6 

1-58 

1 19-3 

8.79 

620.8 

1. 8l 

47-7 

19.36 

524.8 

2.14 

23.8 

32.51 

477-1 

2-15 

4.8 

70.67 

357-8 

3-II 

The  loss  of  oxygen  does  not  become  marked  until  comparatively 
low  pressures  are  reached. 

Carbon  Monoxide  Hemoglobin.  When  a  current  of  carbon 
monoxide  is  led  into  a  blood  solution  it  displaces  the  oxygen  and  forms 


THE   BLOOD.  1 85 

a  very  stable  compound.  One  molecule  of  the  monoxide  takes  the  place 
of  the  molecule  of  oxygen  combined  as  oxyhemoglobin.  This  reaction 
is  accompanied  by  a  change  of  color,  not  as  marked,  however,  as  the 
change  from  reduced  to  oxyhemoglobin.  The  combination  with  car- 
bon monoxide  is  the  reaction  which  takes  place  in  cases  of  poisoning 
with  illuminating  gas,  which  contains  10  to  25  per  cent  of  the  monox- 
ide. The  addition  of  pure  air  does  not  displace  the  combined  gas 
except  where  a  great  excess  is  used. 

Experiment.  Lead  a  current  of  illuminating  gas,  best  the  so-called  "  water  gas," 
into  50  cc.  of  blood  in  a  flask.  Continue  the  passage  of  the  gas  until  a  distinct 
cherry  red  color  is  produced.  When  the  combination  appears  to  be  complete  treat 
a  few  cc.  of  the  liquid  with  Stokes'  solution,  which  fails  to  effect  a  reduction.  With 
portions  of  the  mixture  further  tests  should  be  made  to  illustrate  methods  of  dif- 
ferentiating between  normal  blood  and  blood  containing  much  monoxide.  The  dif- 
ferentiation in  each  case  depends  on  the  greater  stability  of  the  monoxide  hemo- 
globin with  the  reagent  in  question. 

Experiment.  Add  some  strong  solution  of  sodium  hydroxide  to  ordinary  blood. 
This  gives  a  brownish  green  precipitate  at  first  and  then  a  red  solution.  Treat 
blood  saturated  with  carbon  monoxide  in  the  same  manner.  This  gives  a  red  pre- 
cipitate and  finally  a  red  solution. 

Experiment.  Dilute  some  of  the  monoxide  blood  with  four  volumes  of  water 
and  to  the  mixture  add  an  equal  volume  of  a  3  per  cent  tannic  acid  solution.  The 
red  color  persists  much  longer  than  it  would  in  the  case  of  a  simple  oxyhemoglobin 
solution,  which  should  be  tried  for  comparison. 

Experiment.  To  about  half  a  cubic  centimeter  of  the  monoxide  blood  add  20 
cc.  of  water  and  10  drops  of  strong  yellow  ammonium  sulphide  solution.  Shake 
thoroughly  and  then  add  enough  dilute  acetic  acid  to  give  a  faint  acid  reaction.  A 
rose  red  color  appears,  while  with  normal  blood  decomposition  products  are  formed 
which  have  a  dirty  gray  color. 

Experiment.  Mix  1  cc.  of  the  monoxide  blood  with  5  cc.  of  basic  lead  acetate 
solution  and  shake  well.  The  mixture  remains  red,  while  with  normal  blood  under 
the  same  conditions  a  brown  color  results. 

Experiments  have  been  carried  out  to  determine  what  portion  of  the 
hemoglobin  must  be  combined  with  monoxide  to  have  death  follow. 
It  appears  that  if  about  half  the  pigment  in  the  blood  is  still  unchanged 
recovery  may  be  expected  by  free  use  of  air.  The  mere  action  of  a 
great  excess  of  air  may  gradually  displace  the  combined  monoxide. 
The  spectroscopic  appearance  of  the  monoxide  hemoglobin  will  be 
referred  to  below. 

Nitric  Oxide  Hemoglobin.  Under  certain  conditions  nitric  oxide, 
NO,  may  be  combined  with  hemoglobin  to  form  a  very  stable  com- 
pound. The  union  takes  place  molecule  with  molecule  and  may  be 
obtained  by  treating  carbon  monoxide  hemoglobin  with  the  nitric 
oxide,  which  has  the  power  of  breaking  up  the  monoxide  combination. 
The  direct  action  of  nitric  oxide  on  oxyhemoglobin  does  not  lead  to 
the  desired  result,  as  oxidation  of  the  NO  follows  and  the  acid  formed 


I  86  PHYSIOLOGICAL    CHEMISTRY. 

destroys  the  hemoglobin.  In  presence  of  urea,  however,  the  direct 
union  is  possible.  The  substance  forms  crystals  like  those  of  oxyhem- 
oglobin and  gives  a  very  similar  spectrum. 

Sulphohemoglobin.  When  hydrogen  sulphide  is  led  into  a  solution 
of  oxyhemoglobin  in  presence  of  air  a  compound  is  formed  which, 
however,  is  not  permanent.  Decomposition  soon  follows  and  a  green- 
ish brown  mixture  results..  With  reduced  hemoglobin  away  from  the 
air  a  true  compound  is  formed  which  gives  a  characteristic  spectrum, 
and  which  is  much  more  stable. 

Other  Combinations.  Several  authors  have  described  other  com- 
pounds formed  by  the  union  of  hemoglobin  and  gases.  Of  these, 
so-called  carbohemoglobins,  acetylenehemoglobin  and  cyanhemoglobin 
are  the  best  known.  These  combinations  are  but  slightly  stable  and 
have  no  special  importance.  As  acetylene  was  formerly  prepared  on 
the  laboratory  scale  it  was  poisonous,  and  this  property  was  assumed 
to  be  due  to  its  action  on  hemoglobin,  which  was  thought  to  resemble 
that  of  carbon  monoxide.  Since  the  manufacture  of  acetylene  from 
calcium  carbide  was  begun  this  notion  has  been  dispelled.  The  action 
of  acetylene  on  the  -blood  is  very  weak.  In  the  early  laboratory 
product  impurities  present  were  probably  responsible  for  the  observed 
effects. 

DERIVATIVES   OF   HEMOGLOBIN. 

Some  of  these  are  practically  identical  with  hemoglobin,  while  others 
are  products  of  complete  decomposition.     The  first  to  be  considered  is : 

Methemoglobin.  Oxyhemoglobin  in  solution  or  in  crystal  form, 
alone  or  in  presence  of  certain  reagents,  shows  a  great  tendency  to  pass 
over  into  this  modification  which  contains  just  as  much  oxygen  as  the 
original,  held,  however,  in  stable  combination.  Under  the  air  pump 
methemoglobin  does  not  give  up  any  oxygen,  while  from  oxyhemo- 
globin nearly  the  whole  of  the  extra  molecule  may  be  abstracted. 
While  this  formation  of  methemoglobin  takes  place  spontaneously  it  is 
greatly  hastened  by  the  action  of  several  substances,  some  of  which  are 
oxidizing  agents,  while  others  are  reducers.  Of  the  oxidizing  sub- 
stances ozone,  potassium  permanganate,  potassium  chlorate,  iodine, 
potassium  f  erricyanide  and  nitrates  have  been  used,  while  such  reducing 
agents  as  pyrogallol,  pyrocatechol,  hydroquinol  and  hydrogen  even, 
acting  on  the  blood  have  brought  about  a  formation  of  the  stable  met- 
hemoglobin. Certain  substances  given  as  remedies  have  the  power  of 
converting  the  oxyhemoglobin  into  methemoglobin.  Amyl  nitrite, 
acetanilid  and  nitrobenzene  may  be  mentioned  here.     The  poisonous 


THE    BLOOD.  1 87 

action  of  large  doses  of  potassium  chlorate  has  long  been  supposed  to 
be  due  in  part  to  the  same  reaction. 

Solutions  of  methemoglobin  are  not  bright  red  but  reddish  brown, 
and  the  crystalline  substance  is  also  brown.  The  color  of  an  alkaline 
solution  is  red,  but  this  is  not  due  to  a  reconversion  into  oxyhemo- 
globin. Certain  reducing  agents  have  the  power  of  gradually  chang- 
ing the  methemoglobin  back  into  oxyhemoglobin  and  then  into  reduced 
hemoglobin.  Ammonium  sulphide  and  Stokes'  reagent  work  in  this 
way.     The  conversion  may  be  followed  by  aid  of  the  spectroscope. 

A  product  known  as  acid  hemoglobin  is  formed  by  the  action  of 
weak  acids  on  hemoglobin.  This  appears  to  be  a  step  in  the  formation 
of  methemoglobin,  the  spectrum  of  which  it  resembles.  With  strong 
acids  decomposition  takes  place  and  hematin  results. 

Hematin.  It  has  been  already  explained  that  hemoglobin  breaks  up 
readily  into  globin,  about  96  per  cent,  and  hematin,  about  4  per  cent. 
This  decomposition  follows,  as  just  mentioned,  by  the  treatment  with 
strong  acids,  and  also  by  various  other  reactions.  The  product  from 
reduced  hemoglobin  is  known  as  hemochromogen,  while  from  oxyhem- 
oglobin oxyhematin  or  common  hematin  is  obtained.  The  relations 
may  be.  thus  illustrated : 

Hemoglobin  {  Jlobin,  (  +  O  =  hematin 

Hemoglobin  j  hem0chroinogen  j  _  Fe  =  hematolin 

(  globin      r  +  HC1  =  hemin 
Oxyhemoglobin  j  hematin  J  _    0    =  hemochromogen 

(^  —   Fe  =  hematoporphyrin 

Hematin  is  usually  obtained  as  an  acid  combination  or  ester.  In  one 
process  frequently  followed  blood  is  warmed  with  an  excess  of  glacial 
acetic  acid.  Crystals  containing  acetic  acid  separate  on  cooling.  In 
another  process  the  hydrochloride  is  obtained ;  in  either  case  the  free 
hematin  is  secured  by  saponification  with  weak  sodium  hydroxide  solu- 
tion.    Several  formulas  have  been  given  for  hematin;  the  one  most 

commonly  accepted  is 

C«H«N«FeO„ 

while  for  hemin  crystals  the  formula 

C32H30N4FeO3HCl 

has  been  gi  ven.  Kiister  has  recently  given  the  formula  C34H34N4Fe05. 
It  is  possible  that  different  analysts  have  obtained,  not  the  same,  but 
closely  related  products.     Hemin  is  secured  in  minute  brownish  crys- 


1 88  PHYSIOLOGICAL    CHEMISTRY. 

talline  plates,  hematin  as  an  amorphous  bluish  black  insoluble  powder. 
The  spectrum,  which  is  important,  will  be  referred  to  later. 

Hematoporphyrin.  This  is  a  derivative  obtained  by  the  action  of 
acids  on  hematin.  In  this  treatment  the  iron  is  removed,  as  illustrated 
by  the  following  reaction,  when  hydrobromic  acid  is  employed  as  the 
decomposing  agent : 

C32H32N4Fe04  +  2H20  +  2HBr  =  2C1GH18N203  +  FeBr2  +  H2. 

The  substance  appears  to  be  related  to  and  isomeric  with  bilirubin. 
The  alkaline  solutions  of  hematoporphyrin  are  deep  red,  the  acid  solu- 
tions incline  to  deep  violet  or  purple.  The  acid  and  alkali  spectra  are 
very  different  and  characteristic. 

The  relation  of  the  blood  coloring  matter  to  the  bile  pigments  is 
illustrated  by  these  formulas : 

C32H32N404Fe hematin 

C32H36N406 hematoporphyrin 

C32H36N406 bilirubin 

C32H3eN4Os biliverdin 

Hematolin  is  the  name  given  to  an  iron-free  compound  obtained  by 
decomposing  hematin  in  absence  of  air. 

Hemochromogen.  This  is  obtained  by  reducing  a  hematin  solu- 
tion with  ammonium  sulphide  or  with  zinc  dust  and  alkali.  It  forms 
a  dark  red  powder  insoluble  in  water  but  soluble  in  alkalies.  The  solu- 
tion exposed  to  the  air  absorbs  oxygen  and  appears  to  regenerate 
hematin. 

Hematoidin  is  a  red-colored  pigment  which  has  been  found  in  old 
blood  extravasations.     It  seems  to  be  identical  with  bilirubin. 

THE  OTHER  PROTEINS  OF  THE  BLOOD. 

In  addition  to  fibrin  and  hemoglobin  blood  contains  serum  albumin 
and  serum  globulin,  which  have  been  described  already  in  a  previous 
chapter.  The  combined  substances  make  up  about  7  per  cent.  They 
may  be  approximately  separated  by  precipitating  the  globulin  from 
blood  serum  by  addition  of  a  large  volume  of  water  and  also  by  salt 
precipitation,  which  may  be  illustrated  in  this  way : 

Experiment.  Prepare  blood  serum  as  free  as  possible  from  corpuscles  as  already- 
shown  and  mix  about  100  cc.  with  an  equal  volume  of  cold  saturated  ammonium 
sulphate  solution.  A  separation  of  the  globulin  follows.  Filter;  the  filtrate  con- 
tains practically  all  the  serum  albumin  which  may  be  coagulated  by  boiling.  The 
albumin  may  be  purified  by  long  dialysis.  To  recognize  the  globulin  in  the  pre- 
cipitate, first  wash  the  latter  with  more  half-saturated  ammonium  sulphate  and 
then  dissolve  in  slight  excess  of  water.     It  may  be  necessary  to  add  a  little  common 


THE    BLOOD.  1 89 

salt  to  assist  in  the  solution.  On  heating  a  portion  of  this  solution  coagulation 
follows.  On  diluting  some  of  it  very  largely  with  water  precipitation  of  the  globu- 
lin takes  place.  From  the  first  water  solution  most  of  the  salts  may  be  separated 
by  long  continued  dialysis. 

Magnesium  sulphate,  added  in  powder  form  to  saturation,  is  sometimes  used  in 
the  place  of  ammonium  sulphate  to  effect  the  separation  of  the  albumin  and 
globulin.  The  reaction  in  both  cases  depends  on  the  fact  that  serum  albumin  may 
be  salted  out  only  with  great  difficulty. 

It  is  an  interesting  fact  that  other  proteins  do  not  appear  to  be 
present  in  the  blood.  The  various  proteins  consumed  as  food  suffer 
peculiar  changes  somewhere  in  the  body  and  are  converted  into  these 
two.  These  in  turn  serve  for  the  preparation  of  the  various  other 
related  bodies  found  in  the  several  tissues  of  the  organism.  Gelatin 
may  be  formed  in  this  way  from  the  proteins  of  the  blood,  but  it  does 
not  appear  that  gelatin  can  replace  other  proteins  as  a  food  since  it  is 
deficient  in  one  of  the  essential  protein  component  groups,  viz. :  the 
tyrosine  group. 

It  is  not  yet  known  how  constant  the  relation  of  serum  albumin  to 
serum  globulin  is  or  whether  this  relation  is  the  same  in  all  kinds  of 
blood.  Egg  albumin  is  not  equivalent  to  serum  albumin  physiolog- 
ically, since  if  injected  into  the  blood  it  appears  soon  unchanged  in  the 
urine.  The  albumins  of  related  animal  species  seem  to  be  nearly  alike, 
but  this  does  not  hold  absolutely  true  for  animals  of  widely  different 
species. 

THE  SUGAR  OF  THE  BLOOD. 

This  is  found  in  the  plasma  and  has  generally  been  assumed  to  be 
glucose,  C6H120G,  although  good  reasons  may  be  assigned  for  the 
assumption  of  other  sugars  as  well.  Ordinarily  the  simple  sugar 
finally  formed  in  the  digestive  process  is  glucose  and  the  possible  pas- 
sage of  other  sugars  into  the  blood  has  commonly  been  overlooked. 
As  the  amount  of  sugar  in  the  blood  is  small,  about  0.15  per  cent  in 
the  mean,  its  certain  identification  is  a  matter  of  extreme  difficulty;  it 
must  be  remembered  that  separation  from  the  large  amounts  of  proteins 
present  must  be  complete  before  any  accurate  identification  of  the 
remaining  trace  of  sugar  may  be  thought  of.  The  older  observers 
depended  almost  solely  on  the  common  reduction  tests  which  are  not 
very  sensitive  in  dealing  with  traces.  Recent  investigators  have  shown 
that  a  left-rotating  sugar  is  present  and  apparently,  also,  pentoses  in 
traces.  As  glucose  and  fructose  yield  the  same  osozone  this  simple 
reaction  cannot  be  applied  to  detect  a  fructose  content.  Occasionally 
small  amounts  of  disaccharides  appear  to  be  present.  Of  these  maltose 
passes  into  dextrose  by  inversion,  while  saccharose  and  lactose  would 


190  PHYSIOLOGICAL    CHEMISTRY. 

be  eliminated  as  such  by  the  kidney.  Glucoronic  acid  in  combination 
is  also  present  and  this  may  be  confounded  with  a  sugar  in  some  of  the 
tests.     More  will  be  found  on  this  point  in  a  following  chapter. 

SALTS  OF  THE  BLOOD. 

The  total  mineral  matters  of  the  blood,  exclusive  of  the  iron  of  the 
hemoglobin,  amount  to  a  fraction  of  one  per  cent  only,  but  still  are  of 
very  considerable  importance.  These  salts  are  largely  the  chlorides, 
phosphates  and  carbonates  of  the  alkali  metals,  the  potassium  salts 
being  most  abundant  in  the  corpuscles,  while  the  sodium  salts  are  most 
characteristic  of  the  plasma.  It  is  believed  that  the  variations  in  this 
salt  content  are  very  small  normally.  The  nearly  constant  osmotic 
pressure  of  the  blood  points  to  this.  Slight  changes  are  speedily  cor- 
rected by  the  kidneys. 

Experiment.  The  presence  of  reducing  carbohydrate  and  salts  in  the  blood  may 
be  demonstrated  in  this  way.  Mix  about  50  cc.  of  fresh  blood  with  300  cc.  of  water 
and  boil  vigorously  a  few  minutes.  A  drop  or  two  of  acetic  acid  may  be  added 
during  the  boiling  to  maintain  a  nearly  neutral  reaction.  Filter  and  divide  the 
filtrate,  which  should  be  perfectly  clear,  into  two  parts.  Concentrate  one-half  to  a 
volume  of  about  10  cc.  and  apply  the  Fehling  test  for  sugar.  Concentrate  the  other 
half  likewise  and  use  portions  for  tests  for  phosphates  and  chlorides.  The  sulphate 
test  usually  fails  with  the  volume  of  blood  taken.  Evaporate  a  small  portion  of 
this  concentrate  nearly  to  dryness  on  a  glass  slide,  allow  what  is  left  to  cool  and 
crystallize.  Sodium  chloride  crystals  may  be  recognized  by  the  microscope.  To 
some  of  the  evaporated  residue  apply  the  flame  test  (with  spectroscope)  for 
potassium. 

GASES  OF  THE  BLOOD. 

The  blood  holds  several  gases  in  loose  combination.  These  are 
principally  oxygen,  nitrogen  and  carbon  dioxide.  Minute  traces  of 
argon  seem  to  be  present  also,  which  like  the  more  abundant  nitrogen 
must  exist  in  a  condition  of  simple  solution.  The  methods  of  accurate 
gas  analysis  as  applied  to  blood  were  developed  by  Lothar  Meyer,  who 
made  a  number  of  determinations  in  blood  from  different  sources. 
These  methods  have  been  further  improved  by  others  who  have  placed 
many  results  on  record. 

The  mean  amount  of  nitrogen  is  about  2  volume  per  cent.  The 
oxygen  and  carbon  dioxide  are  variable.  In  arterial  blood  the  oxygen, 
which  is  held  mainly  through  the  agency  of  hemoglobin,  amounts  to 
about  2,2  per  cent  by  volume ;  that  is,  from  100  cc.  of  arterial  blood  22 
cc.  of  oxygen  in  the  mean  may  be  obtained  by  aid  of  the  vacuum  pump. 
The  venous  blood  always  contains  less  oxygen,  probably  not  over  15 
per  cent  by  volume.  These  amounts  are  far  larger  than  could  be 
absorbed  from  the  air  through  the  partial  pressure  of  the  oxygen  pres- 


THE    BLOOD.  I9I 

ent.  In  fact  it  may  be  shown  that  only  a  fraction  of  i  volume  per 
cent  may  be  held  by  the  blood  plasma  perfectly  free  from  corpuscles. 

The  loosely  combined  carbon  dioxide  may  vary  from  30  to  40 
volume  per  cent  in  the  arterial  blood,  while  in  venous  blood  it  is  much 
higher,  reaching  nearly  50  volume  per  cent  in  the  mean.  This  gas  is 
held  partly  in  the  form  of  bicarbonate  and  partly  through  the  agency 
of  the  proteins,  especially  the  hemoglobin.  Most  of  the  carbon  dioxide 
is,  however,  held  by  the  serum  and  may  be  largely  drawn  out  by  aid 
of  the  vacuum  pump.  On  withdrawal  of  the  gas  other  weak  acid 
bodies  are  able  to  take  its  place  in  alkali  combination.  It  is  held  by 
some  observers  that  the  globulins  have  this  power.  In  acid  intoxica- 
tions where  mineral  or  organic  acids  increase  in  the  blood  the  carbon 
dioxide  rapidly  decreases  and  may  fall  to  a  tenth  or  twentieth  even  of 
its  usual  value.  These  stronger  acids  take  the  alkali  and  there  is  there- 
fore nothing  left  to  hold  the  carbon  dioxide. 

Some  of  these  points  will  be  taken  up  in  a  later  chapter  in  discussing 
respiration  phenomena. 

Other  Substances.  Besides  the  substances  mentioned  above  the 
blood  always  contains  a  number  of  other  bodies  of  more  or  less  impor- 
tance. Among  these  may  be  mentioned  the  fats,  soaps,  cholesterol, 
lecithin  and  jecorin.  The  total  fats  amount  ordinarily  to  about  0.2 
per  cent,  but  after  a  meal  may  be  temporarily  much  increased.  Minute 
traces  of  fatty  acids  as  soaps  may  be  also  present.  Cholesterol  appears 
to  be  present  in  free  form  in  traces  and  also  as  an  acid  combination  or 
ester.  Lecithins  are  present  in  very  small  amount  in  both  corpuscles 
and  plasma,  but  anything  like  a  quantitative  determination  does  not 
appear  to  be  possible.  The  name  jecorin  is  applied  to  a  peculiar  sub- 
stance containing  phosphorus,  described  by  several  observers  as  occur- 
ring in  blood.  It  is  soluble  in  ether,  like  lecithin,  and  seems  to  exist 
in  combination  with  a  carbohydrate  group  or  similar  reducing  residue. 
The  substance  has  never  been  obtained  in  form  pure  enough  for 
analysis,  and  it  is  even  possible  that  it  may  be  a  mixture  of  several 
compounds,  one  of  which  is  a  combination  of  glucuronic  acid. 

Variations  in  Disease.  In  disease  the  normal  proportions  of  the  various  sub- 
stances may  suffer  marked  changes.  A  decrease  in  the  normal  number  of  corpuscles 
(about  5  millions  to  the  cubic  millimeter  for  men,  4  to  4.5  millions  for  women)  may 
follow  to  the  extent  of  10  per  cent  or  more  in  certain  anemic  conditions.  There  may 
also  be  a  change  in  the  proportion  of  hemoglobin  without  a  change  in  the  number  of 
corpuscles.  The  methods  of  estimating  the  amount  of  hemoglobin  will  be  given 
later.  The  salts  in  the  blood  suffer  a  percentage  decrease  after  consumption  of  large 
quantities  of  water,  but  only  temporarily.  An  actual  decrease  may  occur  in  cholera 
and  inflammatory  diseases.  Tlie  normal  minute  amount  of  sugar  is  increased  in 
diabetes,  but  not  greatly,  because  of  the  eliminating  power  of  (he  kidneys.     It  may 


I92  PHYSIOLOGICAL    CHEMISTRY. 

be  temporarily  increased  by  the  use  of  certain  drugs  such  as  curare,  amyl  nitrite, 
chloral,  or  by  inhalation  of  chloroform  vapor.  After  meals  rich  in  fats  there  is 
a  temporary  increase  of  fat  in  the  blood,  but  a  persistent  increase  is  noticed  in  the 
blood  of  drunkards  and  of  corpulent  individuals.  In  diseases  where  there  is  rapid 
breaking  down  of  proteins  there  is  usually  observed  an  increase  of  fat. 

A  loss  of  blood  to  the  extent  of  one-third  is  not  necessarily  dangerous  if  it  be 
withdrawn  slowly.  If  one-half  the  blood  is  lost  there  is  great  danger  of  death. 
Blood  may  be  added  by  transfusion,  but  for  safety  should  be  from  an  animal  of  the 
same  species.  The  serum  of  one  animal  has  usually  a  destructive  action  on  the 
corpuscles  of  another.  Transfused  blood  then  may  be  a  source  of  danger  rather 
than  a  means  of  saving  life.  This  peculiar  action  of  serum  will  be  referred  to  later 
in  some  detail. 


CHAPTER    XII. 

THE  OPTICAL  PROPERTIES  OF  BLOOD.    USE  OF  THE  SPECTRO- 
SCOPE AND   OTHER   INSTRUMENTS. 

Solutions  of  hemoglobin  and  the  various  modifications  and  deriva- 
tives described  in  the  last  chapter  absorb  light  from  certain  regions  of 
the  spectrum.  The  character  and  extent  of  this  absorption  are  such 
as  to  afford  a  ready  means  of  identifying  blood  or  its  pigments  through 
the  aid  of  the  spectroscope. 

THE  SPECTRUM  FIELD. 

The  absorption  spectra  with  which  we  are  here  concerned  are  all 
found  in  the  middle  portions  of  the  spectrum  between  the  Fraunhofer 
lines  C  and  F,  that  is  in  a  region  easily  observed.  For  practical  pur- 
poses an  elaborate  instrument  is  not  necessary.  Excellent  service  is 
rendered  by  many  of  the  smaller  direct  vision  spectroscopes.  For 
quantitative  tests,  however,  much  more  complete  apparatus  is  required. 
The  blood  spectrum  differs  from  that  of  all  other  red  solutions  and  is 
very  easily  recognized  with  a  little  practice.  As  the  absorptive  power 
of  hemoglobin  is  very  great  dilute  solutions  only  are  used  and  these 
must  be  observed  in  a  rather  shallow  cell,  preferably  in  one  with  par- 
allel sides  about  I  centimeter  apart.  For  illumination  a  good  oil  lamp 
flame  is  excellent ;  a  steady  gas  flame  may  also  be  employed. 

The  general  arrangement  of  the  essential  parts  of  the  spectroscope  is  shown 
by  the  following  figures.  Fig.  12  illustrates,  diagrammatically,  the  path  of  the 
light  rays  through  the  instrument.  From  the  source  F  the  light  enters  the  colli- 
mater  tube  through  a  narrow  slit  and  reaches  the  prism  P,  where  it  suffers  refrac- 
tion and  dispersion.  Beyond  the  prism  the  rays  are  received  by  the  double  con- 
vex lens  of  the  ocular  tube  and  thrown  to  the  eyepiece  at  E.  A  magnified  virtual 
image  is  formed  as  shown  by  the  dotted  lines.  The  third  tube  carries  a  scale,  the 
image  of  which  is  reflected  into  the  ocular  and  shows  with  the  spectrum.  In 
absorption  spectrum  analysis,  with  which  we  are  concerned  here,  the  light  at  F 
must  be  white  and  between  this  and  the  collimator  slit  a  cell  must  be  placed  to 
hold  the  colored  solution  or  diluted  blood.  This  is  shown  in  the  next  figure,  where 
B  is  an  ordinary  kerosene  lamp  with  flat  wick.  The  edge  of  the  flame  is  turned 
toward  the  absorption  cell  and  slit.  The  apparatus  here  figured  is  arranged  for 
absorption  analysis  and,  with  parts  to  be  described  later,  may  be  used  for  quanti- 
tative work. 

For  most  simple  blood  examinations  the  small  direct  vision  spectroscope  shown 
below  may  be  used.  With  proper  combination  of  crown  and  flint  glass  prisms  it 
is  possible  to  practically  correct  the  refraction  and  leave  a  field  with  satisfactory 
dispersion. 

H  '93 


194 


PHYSIOLOGICAL    CHEMISTRY. 


Variation  in  Spectra  by  Dilution.     In  all  dilutions  the  positions 
of  the  absorption  bands  remain  the  same,  but  their  density  and  width 


Fig.  12.     Diagram  of  simple  spectroscope. 


vary  with  the  concentration.     In  a  relatively  strong  blood  solution,  for 
example,  there  appears  to  be  but  one  broad  oxyhemoglobin  band  be- 


Fig.  13.      Spectroscope  arranged  for  absorption  analysis. 

tween  D  and  E,  but  on  proper  dilution  this  breaks  up  into  two  charac- 
teristic bands.     The  question  of  dilution  is  therefore  important  and 


A» 


Fig.  14.      Direct-vision  spectroscope. 


for  any  given  instrument  and  light  the  observer  should  settle  this  by 
a  few  preliminary  experiments. 


THE  OPTICAL  PROPERTIES  OF  THE  BLOOD.  195 

Spectrum  of  Oxyhemoglobin.  This  consists  of  two  bands  in  the 
yellowish  green  portion  of  the  spectrum  between  D  and  E.  The  bands 
have  not  the  same  width,  the  one  near  E  being  slightly  broader  than  the 
other.  The  preparation  of  proper  dilutions  may  be  illustrated  in  this 
way: 

Experiment.  Measure  out  5  cc.  of  defibrinated  blood  and  dilute  it  accurately 
with  120  cc.  of  water.  Filter  into  a  clean  flask  and  use  the  clear  filtrate  for  tests 
to  follow.     Mark  this  mixture  Solution  No.  I. 

Dilute  50  cc.  of  No.  I  with  50  cc.  of  water  and  mark  the  mixture  Solution  No.  II. 
and  continue  this  until  seven  dilutions  in  all  are  secured,  the  first  one  being  I  in 
25,  as  above,  and  the  last  1  in  1600.     This  is  almost  colorless. 

Take  seven  test-tubes  of  thin,  colorless  glass,  and  as  uniform  as  possible  in 
diameter.  Number  them  1  to  7  and  two-thirds  fill  each  one  with  the  dilute  blood 
solution  corresponding  to  its  number.  Place  each  tube  before  the  narrow  slit  of 
the  spectroscope  and  adjust  the  flame  of  an  oil  or  gas  lamp  so  that  its  light  may 
pass  through  the  solution  into  the  slit.  Pull  out  the  draw  tube  until  the  light  is 
properly  focused  and  observe  that  the  bright  field  is  traversed  by  two  black  bands 
which  cut  out  portions  of  the  yellow  and  green.  With  strong  blood  solutions  all 
light  except  red  is  shut  out,  but  with  solutions  of  the  dilutions  2  to  7  the  field  is 
obscured  only  by  the  two  bands.  In  solution  No.  2  they  are  very  dark  and  well 
defined.  With  increasing  dilution  they  grow  fainter  and  are  scarcely  visible  in 
solution  No.  7.  In  all  the  solutions  examined  note  the  position  of  these  bands 
with  reference  to  the  characteristic  colors.  Note  also  that  the  bands  grow  narrower 
with  increasing  dilution,  and  that  it  becomes  more  and  more  difficult  to  locate  the 
edges  of  the  bands  sharply.  This  fact  has  some  bearing  on  questions  of  quanti- 
tative determinations  to  be  referred  to  later.  Some  of  the  common  absorption 
spectra  are  illustrated. 

With  instruments  furnished  with  a  simple  scale  it  soon  becomes  an 
easy  matter  to  fix  approximately  the  limits  between  which  each  band  is 
found  and  also  the  point  of  deepest  absorption  in  each  band.  These 
data  may  be  expressed  in  arbitrary  scale  divisions,  in  fractions  of  the 
distance  from  D  to  E,  or  most  definitely,  in  wave  lengths  of  light,  if 
the  instrument  has  been  graduated  in  that  way.  In  all  accurate  com- 
parisons of  spectra  some  such  method  of  recording  the  observations 
must  be  adopted. 

Spectrum  of  Reduced  Hemoglobin.  It  was  pointed  out  in  the 
last  chapter  that  the  spectrum  of  reduced  hemoglobin  is  very  different 
from  that  of  the  ordinary  oxyhemoglobin.  In  place  of  two  bands  we 
have  after  reduction  a  single  broad  band  filling  three  fourths  of  the 
space  between  D  and  E.  This  is  the  simple  effect  observed  with 
Stokes'  solution.  If  ammonium  sulphide  is  employed  in  place  of 
Stokes'  solution  the  same  broad  band  appears  and  in  addition  a  single 
narrow  band,  the  center  of  which  is  in  the  red  to  the  left  of  D.  This 
narrow  band  may  be  due  to  some  sulphohemoglobin  formed  at  the 


196 


PHYSIOLOGICAL    CHEMISTRY. 


same  time.     It  will  be  recalled  that  the  reduced  hemoglobin  solution 
is  purplish  red  in  place  of  deep  bright  red. 

Experiment.  To  a  dilute  solution  of  blood,  about  1  part  to  50  of  water,  add  a 
few  drops  of  strong  ammonium  sulphide  solution  and  warm  gently  in  a  test-tube 
until  the  change  of  color  noted  above  is  reached.     Now  place  the  tube  before  the 


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slit  of  the  spectroscope  and  observe  the  bands  referred  to,  especially  the  narrow 
one  in  the  red.     Hydrogen  sulphide  gives  practically  the  same  result. 

Experiment.  Repeat  the  above  experiment,  using  Stokes'  solution  instead  of  the 
sulphide.  A  single  broad  band  appears  now;  if  the  liquid  is  shaken  briskly  the  air 
acts  on  the  reduced  coloring  matter  with  oxidizing  effect,  as  shown  by  a  division 


THE    OPTICAL    PROPERTIES    OF    THE    BLOOD.  197 

of  the  band,  but  only  temporarily.  On  standing  a  short  time  the  single  broad  band, 
not  very  sharply  defined,  returns.  For  these  tests  it  would  be  well  to  employ 
several  dilutions,  beginning  with  No.  II  of  the  series  given  above. 

Spectrum  of  Carbon  Monoxide  Hemoglobin.  It  has  been  already- 
mentioned  that  the  spectrum  of  carbon  monoxide  hemoglobin  is  very- 
similar  to  that  of  oxyhemoglobin.     This  maybe  found  by  a  simple  test. 

Experiment.  Into  diluted  blood,  as  before,  pass  a  stream  of  common  illuminat- 
ing gas  until  the  liquid  is  saturated,  which  requires  but  a  few  minutes.  On  placing 
the  tube  in  front  of  the  spectroscope  the  two  dark  bands  described  will  be  seen,  and 
slightly  farther  from  the  yellow  than  is  the  case  with  oxyhemoglobin. 

These  bands  do  not  change  in  extent  or  position  by  agitation  of  the  liquid  with 
air,  as  follows  with  reduced  hemoglobin,  and  when  the  liquid  is  treated  with 
ammonium  sulphide  or  with  Stokes'  reagent  no  reduction  takes  place.  The  two 
bands  persist. 

Spectrum  of  Methemoglobin.  This  is  characterized  by  a  band  in  the  yellowish 
red  and  by  a  broad  band  in  the  blue.  To  some  moderately  dilute  blood  add  a  few 
drops  of  fresh  solution  of  potassium  ferricyanide.  The  mixture  becomes  brown.  It 
has  been  already  explained  that  by  careful  reduction  with  a  small  amount  of  am- 
monium sulphide  hemoglobin  is  regenerated.  This  may  be  followed  by  the  spec- 
troscope. Add  a  few  drops  of  the  sulphide  solution,  allow  the  mixture  to  stand  a 
short  time  and  then  shake  vigorously  in  the  air.     Oxyhemoglobin  bands  now  appear. 

The  Hematin  Spectra.  Of  these  the  spectrum  of  the  pigment  in  weak  acid  mixture 
is  the  most  characteristic.  This  may  be  obtained  by  first  coagulating  10  cc.  of  blood 
and  50  cc.  of  water  by  vigorous  boiling,  enough  weak  sulphuric  acid  being  added 
to  maintain  an  acid  reaction.  The  coagulum  is  separated,  pressed  dry  and  rubbed 
up  in  a  mortar  with  25  cc.  of  absolute  alcohol  and  1  cc.  of  strong  sulphuric  acid 
gradually  added.  The  mixture  is  then  transferred  to  a  flask  and  heated  half  an 
hour  on  the  water-bath.  After  cooling  the  filtered  liquid  may  be  examined.  A 
strong  dark  band  in  the  red  is  plainly  seen. 

Different  spectra  are  found  after  alkaline  treatment.  Warm  some  diluted  blood 
with  a  few  drops  of  sodium  hydroxide  solution  until  a  brownish-green  color  results. 
The  absorption  spectrum  of  this  liquid  is  not  characteristic.  The  whole  of  the 
field  from  red  to  violet  is  dark.  On  careful  reduction  with  a  little  ammonium 
sulphide  or  Stokes'  solution  the  spectrum  of  hemochromogen  or  reduced  hematin 
may  be  obtained.  This  consists  of  two  sharp  bands  between  D  and  E,  somewhat 
like  those  of  oxyhemoglobin,  but  nearer  E. 

QUANTITATIVE  SPECTRUM  ANALYSIS. 

The  amount  of  hemoglobin  in  a  solution  may  be  very  accurately  estimated  by 
the  aid  of  the  spectroscope,  but  an  instrument  with  special  attachments  for  the 
purpose  is  required.  Several  distinct  methods  have  been  applied  for  the  purpose, 
but  the  methods  now  followed  involve  a  simple  direct  comparison  between  light 
which  has  been  weakened  by  passing  through  a  blood  solution  and  light  passing 
into  the  spectroscope  directly.  Such  a  comparison  is  easily  made  by  means  of 
instruments  with  double  collimator  slit,  as  first  introduced  by  Vierordt.  The 
arrangement  of  the  apparatus  made  by  Kruess  is  shown  in  Fig.  13,  while  the 
double  collimator  slit  and  ocular  and  reading  scale  are  shown  in  Fi^s.  16  and  17. 

The  method  of  measurement  depends  on  the  principle  that  there  is  a  simple 
relation  between  the  amount  of  light  absorbed  by  a  solution  and  t lie  concentration, 


198 


PHYSIOLOGICAL    CHEMISTRY. 


that  is  the  number  of  absorbing  molecules  in  the  same.     By  finding  therefore  the 
fraction  of  the  original  light  absorbed  we  can  arrive  at  the  amount  of  absorbing 

substance  in  solution.  The  loss  of  light  in 
passing  through  solutions  of  increasing 
concentration  follows  the  law  worked  out 
by  Lambert  for  the  loss  in  passing  a  series 
of  glass  plates  of  same  thickness  and  color. 
Each  new  layer  absorbs  the  same  fraction 
of  the  light  reaching  it,  and  in  the  same 
way  each  unit  of  added  concentration  ab- 
sorbs the  same  fraction  absorbed  by  the 
first   unit. 

Supposing  the  increased  absorption  of 
light  to  follow  through  the  addition  of  new 
layers  of  absorbing  substance,  the  relation 
between  the  original  and  residual  inten- 
sities may  be  reached  in  this  manner. 
Calling  the  original  intensity  /  and  the  in- 
Fig.  16.  Symmetrical  double  slit  for  tensity  after  passing  the  first  layer  (or 
the  absorption  spectroscope.  first  unit  of  concentration)   /'  we  have 


I'  =  I- 


the  original  intensity  being  reduced  to  i/n  by  the  first  layer, 
and  following  layers  we  have 


By  a  second,  third 


n     n  n     n     11 


7-1. 

nm 


The  last  expression  shows  the  intensity  after  passing  m  layers.     For  purposes  of 
calculation  this  can  be  put  in  another  form,  taking  the  original  intensity  as  unity : 


P 


gives  log  F  =  —  m  log  11.      log  n  = 


log/' 


Fig.  17.     Ocular  and  reading  scale  of  the  Kruess  spectrophotometer. 


THE    OPTICAL    PROPERTIES    OF    THE    BLOOD.  1 99 

In  comparing  the  light-absorbing  powers  of  solutions  some  arbitrary  basis  must 
be  taken.  Practically  the  thickness  of  layer  which  will  reduce  the  original  in- 
tensity to  tV  its  value  is  so  taken.  The  light-extinguishing  power  of  a  substance 
or  its  coefficient  of  extinction,  has  been  defined  as  the  reciprocal  value  of  the 
thickness  of  a  layer  of  the  substance  necessary  to  reduce  the  intensity  of  the 
transmitted  light  to  tb  its  original  value. 

Representing  the  extinction  coefficient  by  E  and  the  reduced  intensity  by  /'  we 
have  from  the  above  formulas : 

log  — 

E  =     and  F  =  — ,     log  n  = =  E. 

m  10  &  I 


E 


Therefore 

E. 


log/" 


In  practice  m  may  be  given  a  constant  value  and  called  I  (the  thickness  of  cell,  for 
example).     The  formula  becomes 

E  —  —  log  r. 

It  was  said  above  that  increasing  the  thickness  of  a  layer  of  absorbing  substance  has 
the  same  effect  as  increasing  its  concentration  in  the  same  degree.  From  this  it 
follows  that  the  extinction  coefficient  must  be  directly  proportional  to  the  con- 
centration. Let  E  and  E'  represent  two  extinction  coefficients  and  C  and  C  the 
corresponding  concentrations,  then 

E:C::E':C. 

The  relations 

E      Ef     E" 
-£>    c?>    -q,  > 

etc.,  must  be  all  equal  and  constant  for  the  same  substance.  This  constant  ratio  is 
a  characteristic  which  connects  the  light-absorbing  power  of  a  solution  with  its 
strength;  it  may  be  represented  by  the  letter  A  and  be  termed  the  absorption  ratio. 
Hence,  for  a  given  color 

The  value  of  the  constant  A  must  be  found  for  a  given  spectrum  region  by  em- 
ploying a  series  of  suitable  concentrations.  The  determination  of  E  consists  in 
finding  the  value  of  the  reduced  light  as  compared  with  the  original ;  from  the 
formula  given  above  the  extinction  coefficient  is  equal  to  the  negative  logarithm  of 
this  diminished  intensity.  In  the  various  forms  of  photometers  employed  in  this 
kind  of  work  the  peculiar  measuring  mechanism  permits  the  direct  and  simple  esti- 
mation of  the  intensity  of  the  light  after  absorption  as  compared  with  the  light 
before  absorption.  We  find  /'  then  as  a  fraction,  and  E  is  the  negative  logarithm 
of  this.  For  example,  suppose  we  have  in  a  liter  0.25  gm.  of  an  absorbing  substance. 
The  concentration,  or  C,  is  0.00025,  which  represents  the  value  per  cubic  centi- 
meter, taken  as  the  unit  of  volume.  Next,  suppose  we  find  with  our  special  meas- 
uring instrument  that  the  value  of  the  light  after  absorption  is  only  0.0436  of  the 
original,  that  is  about  one  twenty-third. 


200 


PHYSIOLOGICAL    CHEMISTRY. 


Substituting  in  our  formula  we  have 


and  finally 


E  =  —  log  /'  =  —  log  0.0436  =  1. 3605 1 


C       0.00025  0 

A  =  —  =  — ^ — -  =  0.000184. 
E       1. 36051 


In  this  way,  by  repeating  the  observations  with  a  number  of  different  strengths  of 
solution  of  the  substances,  we  find  the  value  of  the  constant.  As  the  individual 
observations  may  differ  a  little  the  mean  must  be  taken.  A  becomes  thus  fixed  once 
for  all  for  a  given  spectral  region,  and  its  value  may  be  employed  to  determine  the 
concentration  of  unknown  solutions  since 

C  =  EA. 

Quantitative  spectrum  analysis  by  absorption  is  based  on  these  very  simple  prin- 
ciples. Blood  or  other  substance  to  be  examined  is  placed  in  a  cell  with  plane 
parallel  sides,  preferably  exactly  1  cm.  apart.  The  cell  should  be  half  filled  and  is 
brought  into  proper  position  in  front  of  a  spectroscope  with  a  double  slit,  the  level 
of  the  liquid  just  reaching  to  the  top  of  the  lower  slit.  The  light  from  the 
illuminating  lamp   enters   the   upper   slit   directly  and   the  lower  one  through  the 


Cfr 

I 

J 

Fig.  18. 


Fig.  19. 


Absorption  cell  and  Schulz  glass  prism  as  used  in  quantitative  analysis  by  absorption. 
The  position  of  the  prism  is  shown  in  Fig.  19. 


colored  liquid.  If  the  two  slit  openings  were  the  same  to  begin  with  the  upper 
one  must  now  be  narrowed  until  the  light  passing  through  it  is  reduced  to  the 
intensity  of  the  light  through  the  blood  and  the  lower  slit.  The  width  of  each 
slit  may  be  measured  on  a  micrometer  screw  head  or  in  some  other  convenient 
way.  In  these  observations  but  a  small  portion  of  the  spectrum  is  brought  into 
the  field  of  view.  The  eyepiece  in  the  spectroscope  is  therefore  furnished  with  a 
screen  which  can  be  opened  or  narrowed  at  will  and  symmetrically,  that  is  from 
both  sides,  so  as  to  expose  some  definite  small  portion  of  the  spectrum.  The 
instrument  should  be  so  constructed  as  to  permit  any  desired  portion  of  the  spec- 
trum to  be  quickly  and  accurately  brought  into  the  field. 

In  place  of  using  a  simple  cell  it  is  much  better  in  practice  to  employ  a  cell  with 
so-called  Schulz  glass  prism.  With  the  simple  cell  the  meniscus  formed  at  the  top 
of  the  liquid  projects  a  broad  dark  band  across  the  field  horizontally,  which  makes 
the  comparison  of  the  upper  and  lower  spectra  very  difficult.     With  the  cell  fur- 


THE    OPTICAL    PROPERTIES    OF    THE    BLOOD. 


20I 


nished  with  a  Schulz  prism  this  difficulty  is  largely  overcome,  but  the  details  of  the 
arrangement  can  not  be  explained  here.  They  will  be  easily  understood  by  use  of 
the  instrument.  When  the  Schulz  prism  is  employed  light  enters  the  upper  slit 
through  i.i  cm.  of  solution  and  the  lower  slit  through  o.i  cm.  of  solution  and  the 
i.o  cm.  of  the  clear  glass  prism. 


Name  of  Substance. 

Spectral  Region. 

K  = 

Oxyhemo- 

Hemo- 

Methemo- 

CO-Hemo- 

Bilirubin  in 

Bilirubin  in 

globin. 

globin. 
O.OOI22 

globin. 

globin. 

Chloroform. 

Alcohol. 

569.3-555-5 

O.OOI33 

0.00260 

O.OOI3I 

549-9-540.0 

O.OOIOO 

0.00150 

O.OOI99 

O.OOII5 

558-1-534-3 

O.OOII3 

O.OOO215 

Soi. 2-494.3 

O.OOOO598 

O.OOOI42 

494.3-486.1 

O.OOOO356 

O.OOOIl6 

486.1-480.6 

0.0000209 

0.0O0IO2 

480.6-474.4 

O.OOOOI48 

O.OOOO842 

474.4-4684 

O.OOOOI26 

0.0000700 

468.4-461.7 

O.OOOOII8 

O.OOOO667 

The  absorption  ratios  for  a  number  of  physiologically  important  substances  are 
shown  in  the  above  table.     The  spectral  regions  are  given  in  the  usual  wave  lengths. 


CLINICAL    METHODS    OF    ESTIMATING    OXYHEMOGLOBIN. 

The  spectrophotometric  estimation  of  oxyhemoglobin  as  described 
above  is  not  simple  enough  for  quick  clinical  determinations,  which 
have  to  be  made  in  the  course  of  daily 
practice  by  medical  men.  Other  forms 
of  apparatus  have  been  devised  for 
this  purpose  and  are  in  common  use. 
In  all  of  these,  comparison  is  made 
between  the  blood  under  examination, 
properly  diluted,  and  a  standard  color 
assumed  to  represent  normal  blood 
correspondingly  diluted.  Some  of  these 
appliances  give  pretty  good  results, 
but  others  are  very  faulty  and  the  val- 
ues they  furnish  quite  untrustworthy. 
In  the  following  pages  several  of 
the  commoner  forms  will  be  briefly 
described. 


Fig.  20.  Fleischl  hemometer, 
showing  divided  cell  for  blood  and 
water  and  reflecting  mirror  to  se- 
cure  uniform   illumination. 


Fleischl's  Hemometer.  This  instrument  consists  essentially  of  a  circular  cell 
with  glass  hottom  divided  by  a  vertical  partition  into  two  equal  compartments 
as  shown  below.  In  one  of  these  the  accurately  diluted  blood  is  placed  in  given 
volume.  The  other  compartment  is  filled  with  pure  water  to  the  same  level.  The 
cell  rests  on  a  stage  below  which  there  is  mounted  a  white  reflecting  mirror  by 
means  of  which  light  may  be  thrown  upward  to  illuminate  the  two  compartments 
of   the  cell   uniformly.     Immediately  under  the   water  compartment  a   long  colored 


202 


PHYSIOLOGICAL    CHEMISTRY. 


glass  wedge  is  placed  in  such  a  manner  that  light  must  pass  through  it  into  the 
water.  By  a  rack  and  pinion  mechanism  this  wedge  may  be  moved  to  the  right 
or  left  under  the  water  cell  so  as  to  bring  a  thinner  or  thicker  portion  of  the 
glass  below  the  water.  The  glass  is  colored  by  means  of  purple  of  Cassius  to 
resemble  diluted  blood  as  nearly  as  possible,  and  the  light  shining  through  it  into 
the  water  imparts  a  more  or  less  perfect  blood  color  to  it.     The  wedge  is  moved 


Fig.  21.  Dare's  hemoglobinometer.  R, 
milled  wheel  acting  by  a  friction  bearing  on 
the  rim  of  the  color  disc ;  S,  case  inclosing 
color  disc,  and  provided  with  a  stage  to  which 
the  blood  chamber  is  fitted ;  T,  movable  wing 
which  is  swung  outward  during  the  observa- 
tion, to  serve  as  a  screen  for  the  observer's 
eyes,  and  which  acts  as  a  cover  to  inclose  the 
color  disc  when  the  instrument  is  not  in  use ; 
U,  telescoping  camera  tube,  in  position  for 
examination ;  V,  aperture  admitting  light  for 
lllumination  of  the  color  disc ;  X,  capillary 
blood  chamber  adjusted  to  stage  of  instru- 
ment, the  slip  of  opaque  glass,  W,  being 
nearest  to  the  source  of  light ;  Y,  detachable 
candle-holder ;  Z,  rectangular  slot  through 
which  the  hemoglobin  scale  indicated  on  the 
rim  of  the  color  disc  is  read. 


Fig.  22.  Horizontal  Sec- 
tion, of  Dare's  Hemoglobin- 
ometer, in  which  the.  arrange- 
ment of  parts  is  clearly 
shown.  L  is  the  standard 
wedge-shaped  color  disc. 
The  blood  is  inclosed  between 
the  glass  plates  O  and  P  and 
is  illuminated  by  the  flame  /. 
The  eye  observes  the  blood 
and  the  color  scale  through 
the  apertures  M  and  M' . 


until  the  liquids  in  the  two  compartments  of  the  cell  appear  to  have  the  same  color. 
The  color  in  a  certain  portion  of  the  wedge  is  intended  to  correspond  to  normal 
blood,  or  blood  with  ioo  per  cent  of  the  normal  oxyhemoglobin,  and  degrees  placed 
at  proper  intervals  along  the  wedge  represent  corresponding  higher  or  lower  per- 
centages. For  example,  if  when  the  colors  in  the  two  compartments  are  matched 
the  reading  on  the  wedge  scale  is  97,  it  means  that  the  blood  contains  color  due  to 
97  per  cent  of  the  normal  or  average  oxyhemoglobin  content. 

This  Fleischl  instrument  is  one  of  the  best  in  principle  but  it  must  be  carefully 
used  to  furnish  good  results.  The  color  of  the  wedge  does  not  correspond  to  blood 
color  unless  a  certain  kind  of  white  light  is  employed.  A  wedge  made  for  candle 
light  cannot  be  used  with  sunlight.     In  addition  to  this  difficulty  the  wedges  them- 


THE    OPTICAL    PROPERTIES    OF    THE    BLOOD.  203 

selves  are  often  at  fault.  They  sometimes  fail  to  produce  a  blood  red  with  any  kind 
of  light. 

Miescher  has  suggested  several  improvements  in  the  Fleischl  instrument  which 
render  it  much  more  accurate.  Readings  may  be  made  from  several  different 
dilutions  from  which  a  mean  value  may  be  taken. 

The  Hemoglobinometer  of  Gowers.  This  instrument  has  been  made  in  several 
forms.  The  construction  is  essentially  this.  Two  narrow  glass  tubes  of  the  same 
diameter  are  used;  one  receives  as  a  standard  a  1  per  cent  solution  of  normal  blood, 
while  the  other  is  graduated  from  below  from  o  to  100  degrees  and  is  intended  to 
receive  the  blood  under  examination.  A  measured  portion  of  this  blood,  usually 
20  cubic  millimeters,  is  poured  into  the  tube  and  diluted  with  distilled  water,  a 
little  at  a  time.  After  each  addition  of  water  the  tube  is  shaken  thoroughly  to 
mix  and  a  comparison  made  with  the  standard  tube.  When  the  colors  are  finally 
the  same,  as  read  horizontally,  that  is  across,  not  down  through  the  tubes,  the 
degree  of  dilution  reached  in  the  graduated  tube  is  noted.  This  indicates  the  per- 
centage of  color  present  as  compared  with  the  standard.  A  blood  which  can  be 
diluted  to  100  degrees  (100  times  the  original  small  volume  taken)  contains  100 
per  cent  of  the  normal  hemoglobin  content  or  is  normal,  while  if  it  can  be  diluted 
to  75  degrees  only,  the  comparison  shows  that  this  blood  contains  but  75  per  cent 
of  the  average  hemoglobin. 

In  place  of  using  blood  as  a  standard  a  gelatin  solution  stained  with  picrocarmine 
or  other  stain  is  frequently  employed.  But  in  time  such  color  standards  always 
fade,  and  an  abnormally  high  result  is  recorded  as  a  consequence. 

Dare's  Hemoglobinometer.  In  this  instrument  the  principle  employed  in  the 
Fleischl  apparatus  is  used,  but  the  comparison  is  made  between  the  colored  glass 
standard  and  undiluted  blood.  The  possible  error  due  to  dilution  is  thus  avoided. 
Some  idea  of  the  apparatus  is  given  by  the  illustrations  above.  A  drop  of  per- 
fectly fresh  blood  is  placed  over  the  opening  in  a  capillary  flat  cell  into  which  it 
is  immediately  drawn,  much  as  a  drop  of  water  is  drawn  in  between  a  slide  and 
cover  glass  not  in  absolute  close  contact,  when  the  water  is  put  on  the  edge  of  the 
cover.  The  capillary  observation  cell  is  mounted  at  the  end  of  an  eyepiece  through 
which  it  may  be  clearly  seen.  A  small  portion  of  the  colored  glass  standard  may 
be  seen  at  the  same  time.  The  blood  cell  and  red  glass  are  evenly  illuminated  by 
a  candle  flame  placed  in  fixed  position  in  front  of  the  apparatus.  The  colored  glass 
standard  is  given  the  form  of  a  circular  disk,  which  may  be  rotated  by  a  screw 
motion.  This  disk  is  beveled  from  one  side  to  the  other,  giving  a  wedge  effect  as 
in  the  Fleischl  apparatus.  The  rotation  of  the  disk  brings  therefore  thicker  or 
thinner  portions  of  the  edge  in  the  field  of  the  eyepiece  along  with  the  cell  holding 
the  blood.  When  the  colors  are  matched  the  corresponding  hemoglobin  value  is 
read  off  on  a  scale.  In  practice  this  instrument  is  somewhat  more  convenient  than 
the  Fleischl.     The  accuracy  is  about  the  same. 

Tallquist's  Chart.  This  consists  of  a  small  book  containing  blank  sheets  of  a 
special  fine  grained  filter  paper  and  a  colored  chart  with  a  number  of  shades 
corresponding  to  blood  stains  with  different  hemoglobin  content.  To  make  the 
test  a  small  drop  of  blood  is  drawn  and  placed  on  one  of  the  sheets  of  filter  paper, 
into  which  it  soaks  and  spreads.  The  color  produced  is  compared  with  one  of  the 
ten  shades  in  the  color  scale. 

The  test  is  extremely  simple,  but  its  accuracy  is  dependent  on  the  accuracy  with 
which  the  colors  in  the  chart  are  printed,  and  their  permanence.  Unfortunately 
the  colors  change  as  the  chart,  in  use,  is  exposed  to  light  and  air. 


CHAPTER   XIII. 

FURTHER  PHYSICAL  METHODS  IN  BLOOD  EXAMINATION.    FREEZ- 
ING   POINT    AND    ELECTRICAL    CONDUCTIVITY.    THE 
HEMATOCRIT. 

OSMOTIC   PRESSURE. 

In  many  of  the  phenomena  of  the  body  the  osmotic  pressure  of  dis- 
solved substances  plays  an  extremely  important  part.  This  is  espe- 
cially true  in  the  study  of  the  blood  as  a  whole,  and  it  is  therefore 
proper  at  this  point  to  enter  upon  a  short  explanation  of  what  is  meant 
by  osmotic  pressure  and  what  its  relations  are. 

Nature  of  Osmotic  Pressure.  Solids  in  solution  exert  a  pressure 
in  all  directions  quite  analogous  to  that  observed  with  gases,  and  in 
general  the  laws  connecting  increase  in  pressure  with  concentration  and 
temperature  are  the  same  as  for  gases.  With  many  solids,  however, 
dissociation  in  solution  or  separation  into  ions  takes  place  and  each 
separate  ion  behaves  as  a  whole  molecule  as  far  as  pressure  is  concerned. 

Some  of  the  simple  effects  of  this  pressure  are  easily  observed. 
When  a  drop  of  a  strong  solution  of  blue  vitriol  is  placed  carefully  on 
the  surface  of  a  weak  solution  of  potassium  ferrocyanide  a  precipitate 
of  copper  ferrocyanide  forms  as  a  sheath  or  membrane  around  the 
vitriol  drop  and  holds  it  in  nearly  spherical  form.  If  the  drop  is  prop- 
erly deposited,  which  requires  some  care,  it  will  gradually  enlarge  by 
the  entrance  of  water,  which  dilutes  the  enclosed  copper  sulphate. 
None  of  the  latter  passes  out  and  the  ferrocyanide  solution  evidently 
does  not  enter  since  no  more  precipitate  forms  within  the  drop.  The 
copper  ferrocyanide  membrane  must  possess  therefore  some  interesting 
properties ;  it  is  permeable  for  water,  it  is  not  permeable  for  either  of 
the  salt  solutions.  Similar  membranes  may  be  made  with  a  number  of 
substances  and  their  impermeability  for  many  salt  or  other  solid  mole- 
cules may  be  shown.  Some  membranes  are  permeable  for  certain  salts 
but  not  for  others.  The  fact  of  the  existence  of  pressure  within  such 
a  membrane  may  be  shown  by  the  following  well-known  experiment, 
in  which  a  copper  ferrocyanide  sheath  or  membrane  is  made  in  a  dif- 
ferent manner. 

Experiment.  Procure  a  small  fine  grained  porous  battery  cell,  about  3  to  4  inches 
long  and  1  inch  in  outside  diameter,  and  clean  it  thoroughly.  This  may  be  done 
by  washing  the  cell  with  water,  then  with  weak  hydrochloric  acid  and  finally  with 

204 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION. 


20t 


water  very  thoroughly.  Close  the  cell  with  a  perforated  rubber  stopper,  pass  a 
glass  tube  through  the  perforation  and  connect  the  outer  end  of  this  with  a  suction 
pump.  On  dipping  the  cell  in  water  or  the  acid  it  may  be  drawn  through  the 
pores  of  the  cell  to  effect  the  cleaning.  When  the  cell  is  clean  it  is  placed  in  a 
potassium  ferrocyanide  solution  containing  about  150  gm.  per  liter  and  solution 
drawn  through  by  means  of  the  pump  until  the  pores  are  thoroughly  filled.  Then 
the  cell  is  washed,  inside  and  out,  with  distilled  water 
and  immersed  in  a  blue  vitriol  solution  containing  about 
250  gm.  per  liter.  A  precipitate  is  thus  formed  within 
the  pores  of  the  cell,  which  is  allowed  to  remain  some 
hours  in  the  solution.  The  cell  is  then  removed, 
washed  with  water  and  is  ready  for  use.  Fill  it  with 
a  5  per  cent  cane  sugar  solution,  close  with  the  rubber 
stopper  and  long  narrow  glass  tube  and  immerse  the 
cell  in  a  beaker  of  distilled  water  the  temperature  of 
which  should  be  the  same  as  that  of  the  sugar  solution. 
After  a  short  time  liquid  begins  to  rise  in  the  glass 
tube  which  serves  as  a  kind  of  manometer.  This  is  in 
consequence  of  the  entrance  of  water  to  the  sugar 
solution.  Sugar  can  not  pass  out  in  the  other  direction 
as  the  precipitate  membrane  is  not  permeable  for  it,  but 
it  is  readily  permeable  for  the  water.  The  sugar  in 
it's  effort  to  pass  out  to  the  water  exerts  a  pressure  on 
the  retaining  membrane,  and  it  is  because  of  this  pres- 
sure that  the  water  is  able  to  enter  the  cell.  The  flow 
of  the  water  continues  until  its  hydrostatic  pressure 
exactly  balances  the  sugar  or  osmotic  pressure.  In 
some  cases  mercury  manometers  attached  to  such  cells 
register  pressure,  of  several  atmospheres. 

The  pressure  actually  observed  in  such  an  apparatus  is  just  short  of  that  required 
to  press  the  solvent,  water,  through  the  membrane  in  the  opposite  direction.  Theo- 
retically it  should  amount  to  22.4  atmospheres  for  a  solution  containing  a  gram 
molecular  weight  dissolved  per  liter,  since  it  has  been  found  that  the  osmotic  pres- 
sure of  a  body  is  the  same  it  would  possess  if  it  existed  in  the  condition  of  a  gas 
at  the  same  temperature  and  in  the  same  volume.  A  gram  molecular  weight  of 
hydrogen  (2.014  gms.),  of  oxygen  (32  gms.),  of  nitrogen  or  other  gas  occupies 
a  volume  of  22.4  liters  under  normal  temperature  and  pressure  conditions.  If  con- 
densed into  1  liter  their  pressure  would  be  22.4  atmospheres.  Experiment  has 
shown  that  a  gram  molecular  weight  of  sugar  or  similar  solid  in  water  to  make 
a  liter  volume  exerts  a  pressure  of  22.4  atmospheres.  In  the  case  of  salts  which 
break  up  into  component  parts  or  ions  the  pressure  becomes  correspondingly  greater. 
In  very  dilute  solutions  a  molecule  of  sodium  chloride,  for  example,  exerts  prac- 
tically double  the  pressure  observed  for  a  molecule  of  sugar.  In  this  dilute  con- 
dition the  component  parts,  or  ions,  of  sodium  and  chlorine  seem  to  exert  a  pres- 
sure corresponding  to  whole  molecules. 

The  above  experiment  is  a  somewhat  crude  one  and  is  intended 
merely  as  an  illustration  of  the  development  of  pressure.  For  accu- 
rate measurements  much  more  elaborate  apparatus  must  be  employed 
and  numerous  precautions  observed.  Practically,  however,  osmotic 
pressure  is  always  measured  by  indirect  methods  to  be  explained  later. 
A  familiar  illustration  of  a  semi-permeable  sheath  or  membrane  is 


Fig.  23.  Apparatus  for 
observing  and  measuring 
osmotic  pressure. 


206  PHYSIOLOGICAL    CHEMISTRY. 

found  in  the  red  blood  corpuscle.  Normally  this  holds  its  hemoglobin 
and  certain  salts  because  it  is  suspended  in  a  liquid  which  has  the  same 
osmotic  pressure.  But  if  the  corpuscles  be  placed  in  pure  water  they 
are  seen  to  swell  and  finally  break  because  of  the  passage  of  water 
through  the  cell  sheath  which  is  not  permeable  for  the  solid  contents. 
By  means  of  the  hematocrit,  as  will  be  explained,  it  is  possible  to  find 
the  average  volume  occupied  by  the  corpuscles  in  a  given  sample  of 
blood.  When  mixed  with  water  or  solutions  with  lower  osmotic  pres- 
sure the  corpuscle  volume  increases ;  in  stronger  salt  solutions,  on  the 
other  hand,  the  individual  corpuscles  shrink  in  size  and  their  total 
volume  becomes  less.  The  hematocrit  may  therefore  be  used  to  measure 
or  compare  osmotic  pressures  in  certain  cases. 

INDIRECT  METHODS.    CRYOSCOPY. 

Although  the  blood  contains  about  20  per  cent  of  organic  substances 
and  about  1  per  cent  of  mineral  matters  its  osmotic  pressure  depends 
largely  on  the  latter.  This  is  because  of  the  simple  fact  that  the  large 
gross  weight  of  organic  matter  represents  relatively  but  a  small  number 
of  molecules,  and  the  actual  pressure  is  measured  by  the  total  number 
of  molecules  or  ions  present.  This  osmotic  pressure  in  health  remains 
practically  constant;  even  after  great  loss  of  blood  when  the  total 
volume  is  restored  by  drawing  on  the  lymph  serum,  although  the  rela- 
tive number  of  corpuscles  may  be  much  reduced,  the  osmotic  pressure 
of  the  new  blood  is  practically  unchanged.  This  is  due  to  the  fact  that 
the  blood  and  lymph  serum  are  isosmotic. 

The  Freezing  Point  Method.  The  measurement  of  the  osmotic 
pressure  of  blood  or  any  other  solution  by  the  direct  method  suggested 
by  the  experiment  given  above  is  extremely  difficult.  Several  indirect 
methods  may  be  followed,  two  of  which  are  in  common  application. 
One  of  these  only  is  suitable  for  the  examination  of  blood.  In  the 
first  of  these  methods  the  elevation  of  the  boiling  point  of  the  solution 
is  observed;  in  the  second  the  depression  of  its  freezing  point.  Com- 
paratively simple  relations  obtain  between  the  three  phenomena.  In  a 
solution  the  tension  of  the  vapor  is  decreased  in  proportion  as  the 
osmotic  pressure  of  the  dissolved  substance  increases  and  more  heat 
must  therefore  be  applied  to  actually  lift  the  atmosphere  or  boil  off 
the  solvent.  For  each  gram-molecule  per  liter  dissolved  this  elevation 
of  the  boiling  point  of  water  is  about  0.520.  This  method,  by  noting 
the  elevation  of  the  boiling  point,  cannot  be  applied  to  blood,  because 
of  its  coagulation,  but  there  is  no  drawback  to  the  method  depending 
on  the  separation  of  the  solvent  by  freezing.     With  increase  in  amount 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION. 


207 


of  salt  or  foreign  substance  dissolved  in  the  water,  the  lower  must  its 

temperature  be  brought  to  effect  a  partial  separation  by  freezing  out 

a  portion  of  the  solvent.     The  lowering  of  the 

freezing  point  is  accurately  proportional  to 

the  number  of  molecules   (or  ions)   present. 

The  molecular  freezing  point  depression  for 

water  is  1.850  ;  that  is,  the  freezing  point  of  a 

solution  containing  one  molecular  weight  in 

grams  of  a  substance,  such  as  sugar  or  urea, 

dissolved  in  a  liter,  is  1.850  below  the  freezing 

point  of  water.     The  osmotic  pressure  of  a 

substance  of  which  1  gram  molecule  per  liter 

is  dissolved  in  water,   is   22.4  atmospheres. 

Therefore  a  freezing  point  depression  of  i° 

C.  corresponds  to  an  osmotic  pressure  of  12. 1 

atmospheres. 

Apparatus.  Various  forms  of  apparatus  have  been 
devised  for  the  experimental  determination  of  freez- 
ing point.  The  Beckmann  apparatus  is  most  com- 
monly employed.  It  consists  essentially  of  a  strong 
test-tube  to  contain  the  substance  to  be  examined. 
This  is  suspended  in  a  somewhat  larger  tube  which 
serves  as  an  air  bath.  The  large  tube  finally  is  sup- 
ported in  a  strong  beaker  or  battery  jar  which  receives 
the  freezing  mixture  to  reduce  the  temperature  of 
the  substance  under  experiment.  The  freezing  mix- 
ture may  consist  of  ice,  water  and  salt,  which  must 
be  stirred  up  frequently  to  maintain  a  uniform  degree 
of  cold.  A  very  delicate  thermometer  passes  down 
into  the  substance  in  the  inner  tube,  which  is  also 
furnished  with  a  stirrer  of  platinum  wire.  The  blood 
or  other  liquid  is  stirred  until  coagulation  begins,  the 
thermometer  being  meanwhile  carefully  watched.  The 
temperature  goes  down  at  first  a  little  below  the 
normal  freezing  point,  because  of  overcooling,  but 
soon  rises  and  remains  stationary.  In  experimenting 
with  aqueous  solutions  a  known  weight  of  pure  water 
is  taken  and  its  freezing  point  with  the  thermometer 
used  is  accurately  found.  Then  the  salt  or  other  body, 
which  has  been  accurately  weighed,  is  added  and  a  new 
determination  made.  While  the  principle  is  simple  the 
details  c;dl  for  some  skill  in  manipulation.  Full  de- 
scriptions of  the  method  may  be  found  in  works  on 
physical  or  organic  chemistry,  as  it  finds  applications 
in  many  directions,  and  especially  in  the  determination 
of  molecular  weight.     The  general  appearance  of  the  simple  apparatus  is  here  shown. 

The  freezing  point  of  normal  human  blood  is  about  — 0.560.     As  a 
reduction  of  [°  corresponds  to  an  atmospheric  pressure  of  12.1  atmos- 


Fig.  24.  Beckmann  freez- 
ing point  apparatus.  D  is 
a  fine  thermometer,  C  the 
containing  jar,  B  the  out- 
side or  air  mantle  tube  and 
A  the  tube  in  which  the 
mixture  to  be  observed  is 
placed.  Two  stirrers  are 
shown  ;  one  for  the  cooling 
mixture  in  the  jar  and  one 
for  the  experimental  mix- 
ture. 


208  PHYSIOLOGICAL    CHEMISTRY. 

pheres,  the  normal  osmotic  pressure  of  the  blood  is  about  6.8  atmos- 
pheres. It  makes  but  little  difference  here  whether  we  consider  the 
whole  blood  or  the  plasma  free  from  corpuscles  and  fibrin.  The  result 
is  mainly  due  to  the  small  molecules  present,  and  these  are  inorganic. 
A  solution  of  20  gms.  of  serum  albumin  in  water  to  make  100  cc. 
would  have  a  freezing  point  of  about  — 0.03 °  ;  the  effect  of  the  other 
proteins  would  be  practically  the  same.  A  solution  of  urea  containing 
10  gm.  in  100  cc.  has  a  freezing  point  of  — 3.08°,  one  of  glucose  with 
10  gm.  in  100  cc.  a  freezing  point  of  — 1.03°,  while  a  solution  of 
common  salt  with  the  same  weight  dissolved  would  show  a  depression 
of  about  50. 

Variations.  This  observed  freezing  point  depression  is  normally 
constant  and  nearly  the  same  for  the  blood  of  all  the  common  animals. 
But  temporary  variations  may  occur.  After  consumption  of  large 
quantities  of  water  it  may  sink  to  —  0.51  °,  while  following  a  meal  rich 
in  salty  food  a  further  depression  to  — 0.620,  or  even  lower,  may  be 
observed.  But  these  changes  are  very  speedily  rectified  through  the 
elimination  of  proper  quantities  of  salts  and  water  by  the  kidneys.  If 
an  examination  of  the  blood  shows  a  greater  depression  than  that 
which  may  be  accounted  for  by  absorption  of  food  constituents  a  failure 
of  some  kind  in  the  functions  of  the  kidneys  is  indicated.  Through 
injury  to  the  mechanism  of  these  organs  the  osmotic  pressure  of  the 
blood  may  rise  to  over  12  atmospheres,  corresponding  to  a  depression 
of  the  freezing  point  of  a  whole  degree  or  more. 

Because  of  these  observed  facts  the  determination  of  the  freezing 
point  of  the  blood  has  become  a  test  of  practical  importance  in  the 
diagnosis  of  disorders  of  the  kidney.  With  proper  facilities  the  ex- 
periment may  be  quickly  made  and  will  serve  to  detect  an  abnormality 
in  the  blood  more  readily  than  this  may  be  accomplished  by  chemical 
analysis.  It  is  customary  at  the  present  time  to  designate  this  freezing 
point  depression  by  A.     Thus,  normally,  for  human  blood 

A  =  —  0.56°. 

ISOTONIC  COEFFICIENT. 

When  a  few  drops  of  blood  are  mixed  with  an  excess  of  salt  solution 
of  a  certain  strength  and  the  mixture  allowed  to  stand  at  rest  the  cor- 
puscles gradually  settle  and  leave  a  colorless  liquid  above.  If  the  same 
volume  of  a  certain  weaker  salt  solution  is  taken  with  the  blood  the 
mixture  after  shaking  is  found  to  leave  no  longer  a  colorless  liquid 
above  the  settled  corpuscles,  but  a  somewhat  reddish  liquid.  This  color 
shows  that  the  corpuscles  have  been  broken  and  that  a  little  of  the 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION.  200. 

hemoglobin  has  escaped.     An  experiment  will  illustrate  the  fact;  it  is 
due  to  Hamburger. 

Experiment.  Prepare  a  series  of  common  salt  solutions  of  the  following  strengths  : 
0.7  per  cent,  0.65  per  cent,  0.60  per  cent,  0.55  per  cenf,  0.50  per  cent  and  0.45  per 
cent.  Measure  out  accurately  20  cc.  of  each  into  test-tubes  and  add  to  each  5  drops 
of  defibrinated  bullock's  blood.  Shake  and  allow  to  stand.  Notice  that  in  some 
of  the  tubes  the  corpuscles  have  settled,  leaving  the  salt  solution  practically  clear 
and  colorless ;  in  others  there  is  color,  which  is  greatest  in  the  tube  with  the  least 
salt.  In  the  tube  with  0.60  per  cent  of  salt  there  should  be  no  color,  while  in  the 
tube  with  the  next  weaker  solution  some  appears.  There  must  be  therefore  some 
solution  between  these  two  in  which  the  corpuscles  just  fail  to  give  up  color.  Ham- 
burger found  this  to  be  one  with  0.58  per  cent  of  salt. 

Osmotic  Tension.  Hamburger  made  a  large  number  of  experi- 
ments of  this  description  and  found  the  limiting  value  of  the  strength 
of  solutions  for  which  no  loss  of  color  follows.  He  spoke  of  these 
solutions  as  being  isotonic,  or  as  having  the  same  osmotic  tension  as 
the  content  of  the  corpuscles.  The  numerical  values  found  bear  a 
close  relation  to  the  molecular  weights  of  the  salts  used.  Thus,  the 
following  values  were  noted : 

Molecular  Weight.  Isotonic  Value. 

NaCl    58.5  0.585  per  cent. 

NaBr   103.0  1.02          " 

Nal  149.9  i-55 

KNO3    101.2  r.oi          " 

KBr    119.1  1. 17 

KI     166.0  1.64 

These  values,  while  isotonic  and  isosmotic  with  each  other,  are  not, 
however,  quite  isosmotic  with  the  blood.  A  common  salt  solution 
having  a  percentage  strength  of  0.9  per  cent  has  practically  the  same 
freezing  point  as  the  blood.  Blood  corpuscles  (human)  in  such  a  solu- 
tion do  not  swell  or  shrink,  consequently  lose  no  hemoglobin.  But  if 
placed  in  weaker  salt  solutions  water  is  gradually  absorbed  to  make  the 
outside  and  inside  osmotic  pressure  the  same.  After  a  time,  however, 
with  decreasing  strength  of  the  salt  solution,  so  much  water  is  absorbed 
that  the  limit  of  strength  of  the  corpuscle  sheath  is  reached  and  a  break 
follows.  The  escape  of  hemoglobin  shows  this  point.  With  salt  solu- 
tions this  break  takes  place  with  practically  corresponding  osmotic  pres- 
sures, but  there  are  many  substances  which  do  not  follow  the  rule  at 
all.  This  is  particularly  true  of  solutions  of  urea,  glycerol,  ammonium 
carbonate,  sodium  carbonate  and  ammonium  chloride.  Even  with 
rather  strong  solutions  of  these  bodies  the  corpuscles  fail  to  hold  their 
hemoglobin.  A  satisfactory  explanation  of  the  abnormal  behavior  of 
these  bodies  is  not  known.  Blood  so  changed  is  said  to  be  lake-colored. 
Following  the  Hamburger  designations  normal  blood  was  said  to  be 
»5 


2IO  PHYSIOLOGICAL    CHEMISTRY. 

hyperisotonic,  since  it  contains  more  than  enough  salts  to  hold  the  cor- 
puscle intact. 

HEMATOCRIT   METHODS. 

It  has  been  shown  above  that  the  red  blood  corpuscles  maintain  their 
normal  volume  in  liquids  which  have  the  same  osmotic  pressure  as  the 
blood.  In  liquids  with  a  lower  pressure  they  swell,  while  in  solutions 
possessing  a  higher  osmotic  pressure  than  the  blood  they  contract. 
The  corpuscles  are  extremely  sensitive  to  such  influences,  and  changes 
in  volume  follow  with  even  very  trifling  changes  in  the  osmotic  pres- 
sure of  a  liquid  with  which  the  blood  may  be  mixed.  The  blood  cor- 
puscle may  be  used  then  as  a  kind  of  indicator  to  disclose  variations  in 
osmotic  pressure,  and  substances  may  be  compared  as  to  the  osmotic 
pressure  they  exert  by  noting  their  behavior  with  the  corpuscles. 

It  would  of  course  be  very  difficult  to  prove  anything  by  measure- 
ments on  a  single  corpuscle,  but  it  is  possible  to  make  the  observation 
on  a  large  volume.  If  blood  is  drawn  up  into  a  narrow  tube  of  capil- 
lary dimensions,  placed  in  a  centrifuge  and  rapidly  rotated  the  cor- 
puscles are  thrown  to  the  outer  end  of  the  tube,  which  must  be  closed 
of  course.  The  volume  occupied  by  the  corpuscles  compared  with  the 
original  blood  volume  may  be  easily  seen. 

Koppe's  Hematocrit.  An  instrument  in  which  such  an  observa- 
tion may  be  accurately  and  easily  made  was  devised  by  Hedin  and 
called  the  hematocrit.  A  special  form  of  this  apparatus  was  con- 
structed by  Koppe  and  is  used  for  the  purpose  of  comparison  of  cor- 
puscle volumes.  The  essential  part  of  the  apparatus,  as  shown  in  the 
figure,  is  a  graduated  capillary  pipette  about  7  cm.  in  length,  which  may 
be  closed  at  both  ends  by  small  metal  plates.  At  the  upper  end  the 
capillary  bore  is  widened  out  so  as  to  form  a  small  mixing  vessel.  The 
pipette  proper  has  a  graduation  of  100  divisions.  By  means  of  a 
syringe  attached  to  the  pipette  by  a  bit  of  rubber  tubing  blood  may  be 
drawn  up  into  the  capillary  and  its  volume  accurately  noted.  The 
pipette  may  be  closed  and  rotated  now  rapidly  in  the  centrifugal 
machine,  which  throws  the  corpuscles  to  the  outer  end.  To  prevent 
coagulation  it  is  best  to  moisten  the  pipette  first  with  a  layer  of  cedar 
oil  which  does  not  interfere  with  the  reading  of  the  blood  volume. 
The  relation  between  blood  volume  and  corpuscle  volume  may  thus  be 
read  off  on  the  graduation.  A  drop  of  similar  fresh  blood  is  next 
drawn  into  the  capillary  and  its  volume  noted;  following  this  some 
solution  is  drawn  in  also,  and  then  with  the  blood  up  into  the  wider 
mixing  part,  where  by  means  of  a  bright,  fine  wire  the  two  liquids  may 
be  stirred  together.     The  plates  are  then  put  on  the  ends  of  the  pipette, 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION. 


21  I 


where  they  are  held  by  springs.  The  pipette  may  be  rotated  as  before 
in  the  centrifugal  machine,  the  rotation  being  continued  until  the 
volume  occupied  by  the  corpuscles  becomes  constant.  By  using  a 
number  of  pipette  tubes  it  is  possible  to  employ  different  mixtures  and 
soon  find  one  in  which  the  corpuscle  volume  remains  normal.  If  a 
series  of  sugar  or  salt  solutions  of  known  osmotic  pressure  are  em- 
ployed, that  of  the  blood  must  be  taken  as  equivalent  to  the  pressure  in 
the  solution  for  which  no  change  in  the  volume  of  the  corpuscles  occurs. 


d. 


Fig.  25.  The  essential  part  of  the  Koeppe  hematocrit.  The  measuring  tube  a  is 
closed  by  two  plates,  b  and  c,  which  are  held  fast  by  the  springs  d.  The  tube  is  filled 
by  means  of  a  peculiar  syringe  shown  at  the  right. 


Conversely  the  apparatus  may  be,  and  is  frequently  employed  to  find  osmotic 
pressures  of  solutions.  The  volume  of  the  corpuscles  is  found  in  some  solution, 
of  cane  sugar  for  example,  which  has  about  the  same  osmotic  pressure  as  the  blood, 
but  which  must  be  accurately  known.  Then  other  solutions  of  a  new  substance  are 
tested  until  two  are  found  which  give  volumes,  one  greater  and  the  other  less  than 
that  with  the  sugar.  A  simple  calculation  will  then  give  the  concentration  of  the 
solution  of  the  substance  under  comparison  which  has  the  same  osmotic  pressure  as 
the  standard  sugar  solution.  The  method  would  naturally  fail  for  any  substance 
which  acts  chemically  on  the  blood  or  which  destroys  the  corpuscles,  such  as  urea 
or  glycerol. 

CLINICAL  USES  OF  THE  HEMATOCRIT. 

On  the  assumption  that  the  volume  occupied  by  the  corpuscles  varies 
with  the  number  of  cells,  attempts  have  been  made  to  use  the  hematocrit 
in  place  of  the  cell  counter.  With  normal  blood  cells  the  relation  is 
practically  constant  and  a  volume  of  50  per  cent  in  the  hematocrit  cor- 
responds very  closely  to  the  average  5,000,000  cells  per  cubic  milli- 
meter. But  unfortunately  where  such  a  simple  method  of  making  a 
blood  cell  count  is  the  most  desirable  it  is  at  the  same  time  the  least 


212  PHYSIOLOGICAL    CHEMISTRY. 

reliable,  since  in  disease  the  corpuscles  do  not  always  retain  their 
normal  size.  A  factor  of  perhaps  greater  importance,  however,  is 
obtained  by  taking  the  ratio  of  the  volume  as  found  by  the  hematocrit 
to  the  corpuscle  count  as  made  by  a  hemacytometer.  With  undiluted 
blood  the  hematocrit  may  be  used  to  determine  whether  or  not  pig- 
mentation has  taken  place.  If  the  corpuscles  are  intact  a  nearly  color- 
less serum  is  secured ;  a  more  or  less  reddish  serum  points  to  disintegra- 
tion of  the  corpuscles. 

THE   ELECTRICAL    CONDUCTIVITY    OF   BLOOD. 

Electrolytes.  It  has  been  found  by  experiment  that  certain  solu- 
tions conduct  the  electric  current  while  others  do  not.  Pure  liquids  do 
not  conduct  at  all,  as  a  rule.  Thus  absolutely  pure  water,  glycerol, 
alcohol,  anhydrous  sulphuric  acid  and  similar  substances  are  practically 
non-conductors.  Solutions  of  many  organic  substances  are  likewise 
non-conductors,  practically.  The  sugars,  for  example,  belong  to  this 
class.  But  organic  acids  and  salts  and  many  so-called  basic  bodies  are, 
like  the  corresponding  inorganic  substances,  conductors.  In  general, 
liquid  conductors  or  electrolytes  are  compounds  which  in  solution  sepa- 
rate or  dissociate  into  component  parts  or  ions  more  or  less  perfectly. 


Fig.  26.  Diagram  of  Wheatstone  bridge  connections.  A  represents  a  cell  or  induc- 
tion coil,  ac  the  bridge  wire,  6"  the  standard  resistance  with  which  comparison  is  made, 
R  the  conductivity  cell  containing  the  substance  under  examination.  In  most  con- 
ductivity experiments  A  is  a  small  induction  coil,  a  telephone,  as  shown,  being  employed 
as  the  current  indicator. 

The  mineral  salts  and  inorganic  acids  and  alkalies  are  in  general  good 
conductors,  as  they  "ionize"  to  a  considerable  degree. 

Blood  serum  has  the  power  of  conducting  the  current  and  mainly 
because  of  its  content  of  salts.  The  proteins  in  absolutely  pure  con- 
dition, salt  free,  are  probably  non-conductors.  Some  of  them,  how- 
ever, because  of  their  acid  character  exist  in  combinations  resembling 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION.  213 

salts  and  these  have  a  weak  conducting  power.  But,  because  of  their 
high  molecular  weight,  the  part  which  this  conductivity  plays  in  the 
total  conductivity  of  the  blood  is  very  small.  As  applied  to  the  blood, 
therefore,  conductivity  measurements  give  us  an  idea  of  the  number  of 
salt  molecules  present,  or  inorganic  concentration. 

In  practice  conductivity  is  the  reciprocal  of  resistance,  which  is  the  factor  actually 
measured.  Resistance  is  expressed  in  terms  of  some  standard  arbitrarily  chosen,  and 
comparisons  are  usually  with  the  "  ohm  "  as  the  final  standard.  The  legal  ohm  is 
the  resistance  of  a  column  of  pure  mercury  106.3  cm.  long  and  1  mm.  in  section  at 


Fig.  27.  Simple  Wheatstone  bridge.  The  bridge  wire  of  exact  length  is  stretched 
over  the  graduated  scale.  The  several  wire  connections  are  made  at  the  binding  posts 
lettered. 

a  temperature  of  o°,  but  for  practical  use  resistance  standards  of  wire  are  employed. 
A  series  of  standard  wire  resistances  running  from  a  tenth,  or  hundredth  of  an 
ohm  even,  to  1000  ohms  or  more  is  generally  employed  in  the  form  of  a  resistance 
"  set "  or  "  box." 

In  dealing  with  solutions  the  unit  of  conductivity  is  taken  as  the  reciprocal  of  the 
resistance  of  a  substance  which,  in  the  form  of  a  column  1  cm.  square  and  1  cm. 
long  (a  symmetrical  cubic  centimeter),  has  a  resistance  of  1  ohm.  That  is,  the  con- 
ductivity, k,  is  measured  in  terms  of  that  of  an  ideal  liquid,  one  symmetrical  cubic 
centimeter  of  which  has  a  conductivity  of  1  between  opposite  faces,  or  which  offers 
between  the  same  faces  a  resistance  of  1  ohm.  The  resistance  of  liquids  is  always 
found  in  small  vessels  of  glass  made  in  different  shapes  and  sizes  according  to  the 
character  of  the  liquid.  Small  platinum  plates  are  mounted  in  the  vessels  and  it  is 
the  resistance  of  the  column  between  these  which  is  measured.  Before  use  the 
resistance  capacity  of  the  vessel  must  be  found.  This  is  done  by  measuring  in  it, 
with  the  plates  in  fixed  position,  the  resistance  of  some  liquid  the  conductivity  of 
which  has  been  previously  determined  by  some  standard  method.  The  data  for 
several  solutions  have  been  very  accurately  determined  and  are  everywhere  used 
for  purposes  of  graduation  of  conductivity  vessels.  With  such  a  standard  liquid 
with  conductivity  k  we  find  in  our  cell  the  resistance,  R.  The  resistance  capacity 
C,  is  given  by  the  relation : 

C  =  Rk. 

That  is,  C  is  the  resistance  which  would  be  found  in  the  vessel  if  it  were  filled  with 
a  liquid  of  unit  conductivity,  and  is  used  as  a  constant  in  all  following  calculations 
with  the  same  vessel  when  we  wish  to  find  «.  R  we  always  find  by  direct  measure- 
ment  in  ohms  and   with   C  known   we  have  now: 

C 
K  =  R 

The  resistance  of  liquids  cannot  be  found  as  is  that  of  a  solid  by  means  of  (he 
Wheat-tone  bridge  combination  and  a  galvanometer,  since  under  such  circumstances 
liquids  suffer  hydrolysis  with  rapid  1  hang<    of  re  (Stance.     In  place  of  tli<'  direct  cur- 


214 


PHYSIOLOGICAL    CHEMISTRY. 


rent  and  galvanometer  Kohlrausch  suggested  the  use  of  a  weak  induction  current, 
with  a  telephone  as  current  indicator.  With  this  arrangement,  which  is  illustrated  by 
the  annexed  diagram,  it  is  possible  to  measure  the  conductivity  of  the  serum  or 
other  liquid,  very  readily;  a  simple  form  of  Wheatstone  bridge  is  shown  also. 

ac  represents  the  graduated  wire  of  the  Wheatstone  bridge,  5"  the  standard  resis- 
tance with  which  comparison  is  made,  R  the  cell  containing  the  serum  or  other  liquid 
under  investigation,  T  the  telephone  which  ceases  to  buzz  when  no  current  passes 
through  it  to  or  from  b.  This  gives  the  "  null  "  point  in  the  combination  and  when 
this  is  found  the  following  proportion  holds : 

ab  :  be : :  S :  R. 

ab  and  be  are  read  off  directly  as  bridge  wire  lengths,  6"  is  the  known  comparison 
resistance.     Hence  the  unknown  cell  resistance  is  given  by 


R  =  S 


be 
ab 


As  5"  in  practice  is  always  taken  as  io,  ioo  or  iooo  ohms  and  ac  is  always  divided 
decimally,  tables  are  constructed  giving  directly  the  value  of  R  for  any  value  of  ab 
read  off.  In  practice  the  cell  R  is  always  kept  at  a  constant  temperature,  as  the 
conductivity  of  liquids  varies  greatly  with  temperature  changes.     To  maintain  this 


Fig.  28.      Simple  form  of 
Kohlrausch   conductivity  cell. 


Fig.  29.  Conductiv- 
ity cell  for  poor  con- 
ductors or  small  quan- 
tities. 


constant  temperature  the  cell  is  usually  immersed  in  a  large  water  thermostat,  so 
constructed  that  it  may  be  readily  controlled.     Forms  of  cells  are  illustrated. 

The  electrical  conductivity  of  urine  is  also  an  important  factor  which  may  be 
found  by  the  same  kind  of  apparatus,  and  which  will  be  discussed  later. 

Value  of  the  Conductivity  for  Blood.  Expressed  in  the  terms  just 
explained,  the  value  of  the  conductivity  of  blood  serum  is  about 
k  =  0.012,  or  expressed  in  another  form  very  convenient  for  calcula- 
tion, 120  X  io-4.  A  good  part  of  this  conductivity  is  due  to  the  sodium 
chloride  present.  If  the  chlorides  be  accurately  determined  by  one  of 
the  usual  methods  of  quantitative  analysis  and  the  proper  conductivity 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION.  21 5 

corresponding  to  this  salt  content  be  calculated,  which  is  possible  with 
a  considerable  degree  of  accuracy,  and  subtracted  from  the  total  or 
observed  conductivity  a  remainder  is  obtained  which  measures  the 
"  achloridic  "  conductivity,  that  is  the  conductivity  due  to  the  sulphates, 
phosphates  and  carbonates  present. 

The  conductivity  of  the  salts  in  the  serum  is  somewhat  less  than  in 
pure  water,  but  it  is  possible  to  make  a  correction  for  this  interference 
of  the  proteins  and  obtain  satisfactory  values.  The  conductivity  deter- 
mination coupled  with  a  few  simple  chemical  tests  gives  probably  a 
better  view  of  the  inorganic  combinations  in  the  serum  than  would  be 
found  by  an  examination  of  the  ash  of  the  blood,  since  the  ash  must 
contain  sulphur  and  phosphorus  salts  resulting  from  the  oxidation  of 
the  organic  compounds  of  these  elements.  The  general  method  of  cal- 
culating conductivities  in  a  mixed  fluid  like  the  blood  will  be  discussed 
under  the  head  of  conductivity  of  the  urine.  The  information  fur- 
nished by  conductivity  measurements  is,  it  will  be  seen,  an  extension 
of  that  furnished  by  the  osmotic  pressure  determinations.  By  a  com- 
bination of  the  two  processes  it  is  possible  to  distinguish  approximately 
between  the  concentrations  of  several  classes  of  molecules  present,  and 
to  follow  variations  in  these  concentrations  rapidly.  As  yet  the  clinical 
value  of  the  method  is  somewhat  uncertain,  however. 


CHAPTER   XIV. 

SOME    SPECIAL    PROPERTIES    OF    BLOOD    SERUM.    BACTERICIDAL 
ACTION.     PRECIPITINS,   AGGLUTININS,   BACTERIOLYSINS, 

HEMOLYSINS. 

SELF  PRESERVATION   OF  THE  BLOOD. 

In  earlier  experiments  on  transfusion  of  blood  to  supply  a  loss 
brought  about  by  excessive  bleeding  it  was  recognized  that  the  added 
blood  sometimes  seemed  to  act  as  a  poison  to  the  individual  to  whom 
it  was  given.  It  was  found  later  that  this  toxic  action  followed  the 
passage  of  blood  from  one  species  of  animal  to  another,  but  that  the 
transfusion  from  man  to  man,  from  dog  to  dog  or  from  rabbit  to 
rabbit  was  not  accompanied  by  the  same  danger.  Such  observations 
were  frequently  made  and  gradually  led  to  the  conclusion  that  the 
plasma  or  serum  of  the  blood  of  each  animal  contains  a  something 
which  has  a  destructive  action  on  the  corpuscles  of  other  bloods  and 
which  may  be  designed  to  protect  the  blood  from  the  action  of  any 
foreign  substance.  Various  theories  have  been  put  forward  to  explain 
this  recognized  property  of  the  serum.  As  yet  our  knowledge  in  this 
field  is  largely  of  the  empirical  order,  and  scarcely  suitable  for  clear 
elementary  presentation.  But  the  importance  of  the  subject  as  thus 
far  developed  is  so  great  that  a  short  chapter  on  what  seems  most  satis- 
factorily established  may  not  be  out  of  place.  The  phenomena  in 
question  are  certainly  chemical  and  from  this  side  must  receive  their 
final  explanation.  Numerous  related  phenomena  are  found  to  call  for 
the  same  kind  of  consideration. 

It  has  long  been  known  that  the  large  white  cells  of  the  blood,  the 
so-called  leucocytes,  have  the  peculiar  power  of  destroying  bacteria  or 
other  foreign  cells  which  find  their  way  into  the  blood  stream.  Hence 
these  corpuscles  have  been  called  phagocytes  or  devouring  cells.  This 
destruction  of  small  invading  organisms  they  seem  to  accomplish  by  a 
kind  of  digestive  process  which  in  a  general  way  may  be  followed 
under  the  microscope.  To  some  extent  the  destruction  of  one  kind 
of  blood  by  another  may  possibly  be  accounted  for  in  this  way.  But 
the  chief  action  is  certainly  of  a  different  character.  While  the  white 
cells  are  in  a  measure  protective  agents,  active  in  destroying  elements 
which  would  be  harmful  if  left  in  the  blood,  it  seems  altogether  likely 
that  the  most  important  conserving  forces  in  the  blood  are  soluble  com- 

216 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  21J 

pounds,  possibly  of  the  nature  of  enzymes.  This  view  has  been  grad- 
ually developed  and  rests  on  a  basis  of  experiment,  and  observation. 

Harmful  foreign  bodies  entering  the  blood  may  be  in  the  nature  of 
cells,  as  of  bacteria,  or  they  may  be  the  poisons  called  toxins  produced 
by  bacteria.  Anything  in  the  blood  which  resists  or  overcomes  the 
force  of  this  invasion  is  called  an  anti  body.  Normal  serum  seems  to 
contain  a  number  of  anti  substances,  which  have  received  different 
names,  depending  on  how  or  against  what  they  act.  Some  are  called 
precipitins,  others  agglutinins,  cytotoxins,  etc.,  which  terms  will  be 
explained  later.  In  addition  to  the  anti  bodies  normally  present  in  sera 
in  variable  amounts,  and  which  confer  a  certain  degree  of  immunity, 
there  may  be  produced  artificially  a  greatly  increased  specific  immu- 
nity against  some  particular  invasion.  It  was  the  discovery  of  this  fact 
which  in  reality  led  to  the  systematic  study  of  the  whole  phenomenon. 

The  castor  oil  bean  contains  a  peculiar  poisonous  principle  known 
as  ricin,  which  if  given  in  relatively  large  doses  is  fatal,  but  against 
which  an  animal  may  be  immunized  by  treatment  with  gradually  in- 
creasing small  doses.  An  experiment  made  by  Ehrlich,  to  whom  much 
is  due  in  this  field  of  investigation,  showed  that  the  serum  of  the  treated 
animal  must  contain  now  a  specific  anti  body  capable  of  neutralizing 
the  physiological  action  of  ricin.  He  found  that  if  the  ricin  poison 
and  the  serum  of  the  immunized  animal  are  mixed  in  vitro  in  certain 
proportion,  and  then  injected  into  a  fresh  non-immunized  animal  no 
toxic  action  follows.  The  serum  of  the  first  or  immunized  animal  has 
acquired  the  property  of  chemically  combining  with  or  in  some  manner 
neutralizing  the  action  of  the  poison.  That  something  akin  to  a  chem- 
ical action  is  here  in  question  is  shown  by  the  fact  brought  out  by 
further  experiments  that  certain  proportions  must  be  observed  in  the 
mixing  of  the  serum  and  toxic  substance  just  as  in  the  complete  neu- 
tralization of  an  acid  by  an  alkali.  It  was  further  found  that  this  com- 
bination may  be  hastened  by  heat  and  retarded  by  cold,  which  is  true 
of  most  chemical  reactions.  The  behavior  is  also  specific;  that  is,  the 
animal  immunized  against  the  castor  bean  poison  is  not  immunized 
thereby  against  other  vegetable  toxic  substances,  as  abrin,  for  example, 
and  the  serum  of  the  animal  will  not,  in  vitro,  neutralize  the  toxic  abrin 
solution. 

The  same  general  condition  has  been  recognized  in  connection  with 
other  immunizations  and  the  characteristically  specific  nature  of  the 
anti  body  produced  in  the  serum  has  been  shown  beyond  question. 
The  anti  bodies  protective  against  diphtheria  have  no  effect  against  the 
toxins  of  tetanus  or  other  disease  and  vice  versa.     With  such  facts 


2l8  PHYSIOLOGICAL    CHEMISTRY. 

established,  inquiry  was  naturally  directed  toward  the  question  of  the 
chemical  nature  of  these  substances  and  to  the  question  of  their  mode 
of  action.  In  the  voluminous  discussions  which  have  been  carried  on 
over  these  points  it  is  not  always  easy  to  distinguish  between  observed 
facts  and  stoutly  maintained  theories. 

GENERAL  CHARACTER  OF  THE  ANTI  BODIES. 

Antitoxins  Proper.  Foreign  harmful  agents  gaining  access  to  the 
blood  may  be  of  several  kinds.  Some  of  these  are  soluble  toxic  com- 
pounds, products  of  cell  action,  which  in  their  behavior  bear  some  rela- 
tion to  strong  alkaloidal  poisons.  Many  of  these  toxins  are  produced 
by  bacteria  in  the  animal  body  during  the  progress  of  disease  and  the 
symptoms  observed  are  often  due  to  the  action  of  these  poisons  rather 
than  to  mechanical  disturbances  brought  about  by  the  bacteria  directly. 
The  toxins  as  soluble  products  have  the  power  of  wandering  with  the 
blood  stream  and  thus  reaching  particularly  vulnerable  or  susceptible 
organs.  The  soluble  serum  constituent  which  is  normally  present  in 
small  amount  or  which  may  be  developed  there  to  neutralize  the  toxin 
in  some  manner  is  called  an  antitoxin  in  the  restricted  sense.  There  is 
reason  to  believe  that  the  two  things  combine  with  each  other  in  a  true 
chemical  union  and  leave  a  soluble  inert  product. 

Precipitins.  The  serum  of  normal  blood  contains  constituents 
antagonistic  not  only  to  toxic  substances  but  to  other  sera  as  well. 
The  serum  of  one  animal  tends  to  precipitate  or  render  cloudy  the 
serum  of  another.  This  effect  may  be  greatly  increased  by  a  kind  of 
cultivation,  which  may  be  illustrated  in  this  way.  Rabbits'  blood  has 
normally  some  antagonism  for  ox  blood,  but  if  sterile  ox  serum  be 
injected  into  the  rabbit,  intraperitoneally  or  intravenously,  beginning 
with  small  doses  and  increasing  through  a  number  of  days,  a  condition 
is  finally  reached  in  which  the  rabbit  blood  serum  shows  a  very  strong 
precipitating  power  for  ox  serum.  The  small  amount  of  anti  body  in 
the  rabbit's  blood  has  evidently  increased  enormously  through  this 
treatment.  The  organism  through  the  attack  of  the  foreign  serum 
gradually  develops  a  protective  agent  which  acts  through  exclusion  or 
precipitation.     This  serum  constituent  is  called  a  precipitin. 

A  vast  number  of  experiments  have  been  made  in  this  field  and  the 
subject  has  importance  in  different  directions.  We  recognize  not  only 
the  normal  effort  of  the  blood  to  protect  itself  in  this  way,  but  also  the 
remarkable  power  of  development  in  the  peculiar  anti  body  here  con- 
cerned. From  another  standpoint,  however,  the  phenomenon  has 
assumed  even  greater  importance  and  that  is  in  the  identification  of 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  219 

blood.  This  precipitin  reaction  like  the  others  is  specific  and  the  serum 
of  the  rabbit  immunized  with  ox  serum  will  react  only  with  the  ox 
serum.  But  precipitins  do  not  seem  to  be  formed  in  the  blood  of 
animals  which  are  closely  related.  The  serum  of  a  rabbit  which  has 
been  treated  with  pigeon  serum  will  not  react  with  chicken  serum;  an 
anti  rabbit  serum  cannot  be  secured  by  treating  a  guinea-pig  with  the 
serum  of  rabbit's  blood.  These  general  facts  have  been  confirmed  by 
many  observations. 

Blood  Tests  with  Serum.  The  method  of  utilizing  these  generalizations  is 
essentially  this.  Rabbits  are  the  animals  commonly  used  for  experiments,  since  they 
bear  the  treatment  in  general  well  and  yield  a  fairly  large  quantity  of  immunized 
blood  later.  Each  rabbit  is  treated  by  injection  with  the  blood  serum  of  one  animal, 
these  injections  being  repeated  a  number  of  times,  through  several  weeks.  Then 
the  rabbit  is  killed,  bled  and  the  blood  allowed  to  stand  for  separation  of  clot. 
The  clear  serum  is  preserved  in  sealed  tubes  for  future  use,  sometimes  with,  some- 
times without  addition  of  an  antiseptic,  as  putrefaction  does  not  appear  to  impair 
the  reaction.  By  immunizing  rabbits  separately  with  the  blood  of  man,  the  ox,  horse, 
pig,  dog,  sheep,  goat,  chicken,  etc.,  a  whole  series  of  test  anti  sera  will  be  obtained, 
with  which  it  is  possible  to  identify  most  of  the  common  bloods.  Not  much  blood 
is  required  in  the  tests.  A  drop  or  two  of  blood,  from  a  dried  clot  for  example, 
is  soaked  in  water  or  normal  salt  solution,  the  liquid  obtained  filtered  to  clarify  it 
and  treated  in  a  small  test-tube  with  two  or  three  drops  of  the  test-serum.  The 
liquid  to  be  tested  need  not  be  strong.  In  practice  it  should  be  divided  into  a 
number  of  small  portions  in  test-tubes  and  each  portion  should  receive  a  few 
drops  of  an  immunized  rabbit  serum.  Precipitation  or  clouding  will  occur  in  the 
tube  to  which  the  corresponding  anti  serum  is  added.  For  example,  if  the  original 
clot  of  blood  was  human  blood  the  extracted  dilute  serum  in  all  the  tubes,  except 
the  one  to  which  rabbit  blood  immunized  with  human  blood  was  added,  will  remain 
clear;  other  tubes  with  portions  of  the  extracted  clot  show  no  reaction  with  the  few 
drops  of  rabbit  sera  immunized  with  the  blood  of  other  animals. 

The  medico-legal  importance  of  this  reaction  has  already  been  recognized  and 
tested  in  many  ways.  The  blood  of  certain  monkeys  seems  to  react  as  does  human 
blood,  but  those  who  have  practiced  the  test  most  testify  as  to  its  certainty  and 
wide  applicability  in  distinguishing  between  hu"man  blood  and  the  blood  of  the 
common  domestic  animals. 

The  Cytotoxins.  This  name  is  given  to  certain  anti  compounds  in 
blood  which  are  destructive  of  form  elements.  The  anti  bodies  before 
considered  deal  with  soluble  substances,  but  here  we  have  to  consider 
something  whose  power  extends  to  the  breaking  down  of  cell  struc- 
tures, whether  of  the  blood  corpuscle  or  of  bacteria.  In  the  one  case 
the  term  hemolysin  is  used  to  describe  the  anti  body;  in  the  other  case 
the  term  bacteriolysin  is  employed.  In  their  mode  of  action  these 
agents  appear  to  be  much  alike  and  both  are  found  in  normal  bloods. 
Both  also  may  be  greatly  increased  artificially. 

The  hemolytic  action  of  one  blood  on  another  was  first  observed  in 
experiments  on  blood  transfusion  which  have  been  referred  to  already. 


220  PHYSIOLOGICAL    CHEMISTRY. 

A  foreign  blood  introduced  into  the  circulation  of  an  animal  of  a  dif- 
ferent species  brings  about  a  variety  of  changes;  clots  are  sometimes 
formed  and  from  resultant  changes  in  pressure  serous  exudations  may 
follow.  Hemoglobinuria  is  a  general  consequence  and  this  of  course 
results  from  a  breaking  down  of  blood  corpuscles  in  quantity.  It  has 
been  assumed  by  some  writers  that  this  hemolytic  effect  is  possibly  due 
to  altered  osmotic  pressure  in  the  blood,  as  similar  phenomena  are 
brought  about  by  the  admixture  of  blood  with  weak  solutions.  But 
the  peculiar  specificity  of  artificial  hemolysis  shows  that  this  explana- 
tion is  not  satisfactory. 

If  the  blood  of  man,  for  example,  receives  an  injection  of  human 
blood  under  proper  condition  no  harm  results,  but  if  ox  blood  be  used 
the  case  is  different.  A  large  transfusion  of  the  ox  blood  might  be 
at  once  fatal,  the  hemolysins  of  that  destroying  the  human  corpuscles. 
On  the  other  hand  transfusion  of  small  amounts  of  ox  blood  would 
have  different  effects  varying  with  the  manner  of  transfusion.  With 
but  little  ox  blood  added  the  human  hemolysins  would  be  greatly  in 
excess  and  by  their  chemical  mass  action  would  bring  about  a  relatively 
great  destruction  of  the  ox  blood  corpuscles,  while  the  corpuscles  of  the 
human  blood  would  suffer  but  little  change.  But  more  than  this  would 
probably  take  place  as  illustrated  by  what  has  been  observed  with  cer- 
tain lower  animals.  The  serum  of  the  eel  is  especially  destructive  of 
the  corpuscles  of  rabbit's  blood  and  a  large  injection  of  eel  serum  into 
the  rabbit  would  produce  death.  With  repeated  small  doses,  however, 
the  rabbit's  blood  is  stimulated  to  develop  the  antitoxin  or  antihemo- 
lysin  which  protects  against  the  eel  serum  poison.  A  condition  of 
immunity  is  thus  reached,  and  what  has  been  shown  for  the  rabbit  has 
been  shown  for  other  animals.  An  explanation  of  the  origin  of  the 
hemolysins  will  be  offered  below,  but  now  we  are  concerned  only  with 
the  fact. 

In  the  above  cited  experiment  the  eel  serum  develops  gradually  an 
antihemolysin  which  works  to  prevent  further  destruction  of  the  rabbit 
corpuscles.  But  the  action  does  not  stop  here.  By  injection  of  blood, 
hemolysins  for  the  corpuscles  of  this  blood  are  also  formed.  Numer- 
ous experiments  of  this  kind  have  been  made  with  animals.  For  ex- 
ample, when  rabbit's  blood  is  gradually  injected  into  the  dog  the  pro- 
duction of  hemolysins  is  stimulated  and  the  serum  of  the  dog  so  treated 
is  found  to  be  far  more  toxic  for  the  rabbit  than  was  the  original 
serum.  This  toxic  action,  by  test-tube  experiments,  has  been  found 
to  be  parallel  to  the  hemolytic  action  of  the  dog  serum  on  the  rabbit 
corpuscles,  thus  showing  that  the  toxicity  may  depend  on  the  destruc- 


SPECIAL  PROPERTIES  OF  BLOOD  SERUM.  221 

tion  of  the  corpuscles.  The  hemolysins  produced  as  just  explained 
are  also  in  general  specific  in  their  character,  which  can  be  followed  by 
experiments  in  vitro  as  well  as  in  corpore. 

In  general  the  bactericidal  action  of  serum  resembles  its  hemolytic 
action,  although  control  experiments  in  vitro  cannot  be  as  readily  per- 
formed. We  have  therefore  the  bad erioly sins  to  consider  along  with 
the  other  cell  destroyers.  These  bodies  exist  to  some  extent  in  normal 
blood  and  other  body  fluids  and  serve  to  protect  the  organism  against 
the  attack  of  bacteria  which  in  any  way  gain  admission  to  the  body. 
Milk  is  relatively  rich  in  bacteriolysins  and  hence  the  well-known 
germicidal  action  which  has  been  long  recognized.  In  this  respect  the 
behavior  of  mother's  milk  is  more  marked  than  that  of  cow's  milk. 
Besides  the  cytotoxins  of  this  class  normally  present  in  blood,  specific 
bodies  may  be  developed  by  the  general  methods  followed  in  other 
cases,  that  is  by  the  gradual  introduction  of  cultures  of  specific  bac- 
teria, beginning  with  cultures  of  relatively  little  virulence.  In  this 
way  the  blood  of  the  treated  animal  becomes  immune  for  some  one 
bacterium  species  and  develops  the  power  of  destroying  that  bacterium 
only  for  which  it  was  specially  immunized.  The  same  animal  may  be 
immunized  against  several  kinds  of  bacteria  at  the  same  time  and  the 
different  specific  bacteriolysins  do  not  appear  to  have  any  destructive 
action  on  each  other.  They  exist  together  in  the  blood  just  as  the 
different  proteins  may  exist  side  by  side. 

Through  the  process  of  immunization  the  blood  of  the  animal 
acquires  not  only  the  power  of  attacking  the  specific  bacterium,  but 
also  the  toxins  of  this  bacterium.  At  least  two  kinds  of  anti  bodies 
are  therefore  produced  and  there  are  conditions  in  which  only  one  of 
these  may  be  active.  A  serum  may  be  active  in  the  breaking  down  of 
bacterial  cells,  but  inert  as  against  the  poisons  produced  by  such  cells. 
The  complexities  of  the  phenomena,  however,  cannot  be  detailed  here. 
It  should  be  said  further  that  bacteria  produce  hemolysins,  which  are 
probably  part  of  the  toxins  secreted.  At  any  rate  some  of  the  toxins 
found  in  cultures  are  strongly  hemolytic. 

Agglutinins.  Among  the  several  modes  of  defense  observed  in 
sera  of  various  animals  that  of  agglutination  of  invading  cells  must 
next  be  briefly  considered.  We  have  seen  that  blood  cells  and  bacterial 
cells  may  suffer  a  kind  of  dissolution  through  the  action  of  hemolysins 
or  bacteriolysins,  and  that  a  foreign  serum  is  attacked  by  the  precipi- 
tins. In  addition  to  these  defensive  anti  bodies  there  are  present  others 
which  work  by  agglutinating  or  precipitating  cells.  A  certain  simi- 
larity exists  between  these  bodies  and  the  precipitins,  but  investigations 


222  PHYSIOLOGICAL    CHEMISTRY. 

appear  to  show  that  they  are  distinct.  The  agglutinating  power  is 
found  in  normal  serum,  and  like  the  other  anti  agencies  it  may  be 
greatly  increased  artificially  and  by  the  same  general  means.  Agglu- 
tinins as  precipitating  agents  enter  into  a  loose  kind  of  combination 
with  the  cells  which  they  throw  down.  There  is  here  a  suggestion  of 
combination  -in  some  kind  of  chemical  proportions. 

Bacteria  agglutinins  and  blood  cell  agglutinins  are  to  some  extent 
specific,  but  apparently  less  so  than  are  the  precipitins.  Because  of  this 
specificity  the  phenomenon  has  been  applied  in  a  method  of  diagnosis. 
Following  observations  of  Gruber  and  others,  Widal  suggested  a  test 
which  is  now  commonly  employed  in  diagnosis  of  typhoid  fever.  It  is 
essentially  this : 

Widal  Test.  A  small  amount  of  the  blood  or  serum  of  the  sus- 
pected typhoid  fever  patient  is  mixed  on  a  slide  with  a  bouillon  culture 
of  typhoid  bacilli.  After  a  time  the  mixture  is  examined  with  the 
microscope.  If  the  suspected  blood  contains  the  agglutinins  developed 
in  the  disease,  "clumping"  or  precipitation  of  the  bacteria  from  the 
culture  must  follow.  (It  is  held  as  characteristic  that  loss  of  motility 
must  also  be  observed.)  The  intensity  of  the  agglutinin  reaction  may 
be  estimated  by  noting  the  degree  of  dilution  in  which  the  blood  serum 
will  still  agglutinate  the  bacteria  from  the  bouillon  culture. 

Opsonins.  There  has  been  much  discussion  as  to  the  manner  in 
which  the  phagocytic  power  of  the  leucocytes,  already  referred  to,  may 
he  increased.  By  one  school  of  observers  it  is  held  that  increased 
phagocytosis  is  due  to  a  modification  in  the  leucocytes  themselves,  which 
modification  is  produced  by  a  variable  element  in  the  serum  in  question. 
The  name  stimulin  has  been  given  to  this  agent  which  is  able  to  increase 
the  activity  of  the  white  cells  in  the  destruction  of  bacteria.  Another 
and  more  generally  accepted  view  is  that  increased  phagocytic  action  is 
due,  not  so  much  to  these  cells  themselves,  but  to  a  peculiar  change  in 
the  bacterial  organisms,  which  are  to  be  destroyed.  Wright  and  others 
following  him  hold  that  the  serum  contains  a  specific  ferment-like  body 
whose  function  it  is  to  prepare  or  so  modify  invading  bacterial  cells 
that  they  may  be  readily  engulfed  and  destroyed  by  the  phagocytes. 
This  specific  agent  is  called  an  opsonin,  and  it  has  been  shown  that  the 
opsonic  power  of  a  serum  is  subject  to  great  fluctuation.  In  disease 
it  may  be  greatly  diminished ;  on  the  other  hand,  it  may  be  artificially 
stimulated  in  certain  directions  so  as  to  develop  a  marked  bactericidal 
action.  From  this  point  of  view  bloods  may  be  compared  through  the 
so-called  opsonic  index,  which,  briefly,  is  the  ratio  of  the  bacteria- 
engulfing  power  of  ioo  washed  white  cells  in  contact  with  the  serum 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  223 

of  a  certain  blood,  to  the  power  possessed  by  ioo  similar  cells  in  contact 
with  the  serum  of  a  normal  blood,  taken  as  a  standard,  the  cells  and 
sera  in  each  case  being  brought  in  contact  with  the  same  number  of 
bacteria.  The  observation  is  made  by  the  aid  of  a  high-power  micro- 
scope. The  opsonic  index  observation  has  become  of  such  importance 
that  it  is  frequently  used  in  diagnosis,  and  the  opsonic  treatment  of 
disease  is  directed  toward  increasing  this  function  or  power  of  the 
blood  of  a  patient,  by  properly  graduated  injections  of  tuberculin,  for 
example,  that  a  higher  index  is  gradually  developed.  It  is  but  fair  to 
state  that  the  doctrine  of  opsonins  has  its  active  critics  as  well  as 
adherents. 

Other  Anti  Bodies.  In  the  above  brief  survey  of  the  subject  of 
anti  bodies  present  or  developed  in  the  blood  only  those  which  have 
been  the  object  of  most  frequent  investigations  have  been  mentioned. 
Bacteriologists  have  called  attention  to  numerous  other  varieties  or  sub- 
divisions, but  it  is  not  the  purpose  of  this  chapter  to  take  up  the  dis- 
cussion of  details.  What  has  been  given  is  sufficient  to  call  attention 
to  the  broader  principles  concerned. 

CHEMICAL  NATURE  OF  THE  ANTI  BODIES. 

On  this  topic  much  has  been  written,  but  as  yet  no  satisfactory 
answer  can  be  given  to  the  question :  What  are  they  chemically  ?  As 
formed  in  the  serum  of  blood  or  in  milk  it  may  reasonably  be  assumed 
that  they  must  bear  some  relation  in  composition  to  the  protein  bodies. 
On  this  basis  attempts  have  been  made  to  separate  them  by  fractional 
precipitation  reactions  such  as  were  developed  by  Hofmeister  and 
others  for  the  proteins  and  which  have  been  detailed  in  former  chapters. 

It  appears  from  the  evidence  thus  far  offered  that  some  of  these 
bodies,  at  least,  must  be  classed  among  the  globulins.  Pick  and  others 
have  recently  been  able  to  separate  the  active  substances  from  several 
kinds  of  immune  sera  and  establish  pretty  accurately  the  limits  of  pre- 
cipitation. The  active  fractions  separated  contained  the  real  anti 
bodies  in  minute  amount  only,  probably.  In  some  cases  they  were 
found  in  the  euglobulin  fraction,  and  in  other  cases  in  the  pseudo- 
globulin  fraction  of  the  precipitate. 

There  has  been  much  speculation  as  to  the  part  of  the  blood  which 
gives  rise  to  these  various  anti  bodies.  They  are  soluble  and  may  not 
be  separated  by  filtration  but  on  dialysis  they  behave  as  other  sub- 
stances of  very  high  molecular  weight.  In  many  respects  they  resemble 
highly  active  proteolytic  ferments  or  enzymes,  as  the  characteristic 
phenomena  are  exhibited  even   in  dilutions  of  the  active  serum  of 


224  PHYSIOLOGICAL    CHEMISTRY. 

I  :  20,000,  and  heat  and  chemical  reagents  interfere  with  the  active 
properties  much  as  in  the  case  of  the  enzymes.  But  there  are  appar- 
ently some  exceptions  which  have  led  certain  authors  to  deny  their 
enzyme-like  character.  From  the  sum  of  the  facts  observed  in  the 
occurrence  and  action  of  the  anti  bodies  several  writers  have  been  led 
to  think  of  them  as  derived  from  the  breaking  down  of  the  highly  com- 
plex polynuclear  white  corpuscles  of  the  blood.  The  behavior  of  these 
in  the  "living"  condition  has  been  already  referred  to;  in  their  disin- 
tegration it  is  possible  they  may  give  off  more  and  more  of  the  groups 
on  which  their  activity  depends.  But  there  are  other  possibilities  and 
these  will  be  referred  to  in  the  following  section. 

ORIGIN  AND  MODE  OF  ACTION  OF  THE  ANTI  BODIES. 
EHRLICH'S  THEORY. 

In  the  early  days  of  observations  on  blood  serum. immunity  the  doc- 
trine of  the  phagocytes  received  considerable  attention.  With  increase 
of  knowledge  this  theory  was  seen  to  be  inadequate  to  account  for 
accumulating  facts,  and  the  assumption  of  the  soluble  proteolytic  fer- 
ments, the  alexins,  was  next  to  attract  attention.  These  may  be  formed 
from  the  polynuclear  leucocytes  or  more  remotely  in  the  organs  where 
these  cells  may  have  their  origin,  in  the  spleen,  for  example,  and  in  the 
marrow  of  bones.  As  the  name  indicates  the  alexins  are  protective 
substances,  but  the  simple  assumption  of  these  bodies  acting  alone,  as 
a  chemical  reagent  would  for  example,  in  the  annihilation  of  intruding 
bacteria  was  soon  seen  to  be  too  narrow  to  accord  with  experience. 
The  phenomenon  of  immunity  through  the  alexins  or  other  bodies  is  a 
complex  one,  but  has  received  much  elucidation  through  numerous 
observations  of  recent  years.  One  of  the  most  important  of  these  is 
concerned  with  the  so-called  Pfeiffer  experiment. 

Pfeiffer's  Phenomenon.  In  his  well-known  experiments  on  the 
behavior  of  cholera  bacteria  Pfeiffer  found  that  the  serum  of  an  animal 
which  had  been  immunized  against  cholera,  when  tested  in  vitro  against 
the  vibrios,  seemed  deficient  in  bacteriolytic  power,  possessing  no 
greater  activity,  evidently,  than  that  due  to  the  proteolytic  ferment  of 
the  normal  serum.  Activity  may  be  given  to  the  immunized  serum, 
however,  by  injecting  it  back  into  the  peritoneal  cavity  of  the  animal; 
cholera  vibrios  injected  at  the  same  time  are  quickly  destroyed.  The 
action  is  strictly  specific,  since  typhoid  bacilli  injected  in  the  same  way 
remain  active.  It  was  found  also  that  the  same  result  can  be  reached 
with  the  vibrios  by  adding,  in  vitro,  fresh  normal  serum  to  the  latent 
immunized  serum.     The  vibrios  succumb  as  they  did  in  corpore,  show- 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  22  5 

ing  that  a  vital  action  is  not  in  play.  Numerous  similar  observations 
were  made  by  Bordet  and  others.  From  all  these  experiments  it  is 
evident  that  at  least  two  things  are  concerned  in  the  bacteriolytic  action 
and  the  analogous  hemolytic  phenomena.  The  immune  body  devel- 
oped in  the  course  of  immunization  does  its  work  as  a  cell  destroyer, 
whether  a  blood  corpuscle  or  a  bacterium,  only  by  the  aid  of  something 
normally  present  in  fresh  serum.  The  specific  immune  body  was  found 
to  be  thermostable,  that  is  it  withstands  a  temperature  of  550— 70 ° 
without  loss  of  its  special  properties.  If  after  being  warmed  to  this 
extent  the  immune  serum  is  cooled  to  below  55 °  and  mixed  with  fresh 
normal  serum  the  full  cytolytic  activity  appears.  On  the  other  hand 
the  ferment  in  the  normal  serum  is  thermolabile ;  a  temperature  of  550 
or  above  destroys  it  permanently.  For  this  thermolabile  normal  fer- 
ment Ehrlich  proposed  the  name  addiment.  This  is  the  same  as  the 
alexin  body.  The  term  complement  is  now  more  commonly  used  to 
describe  the  same  thing  which  seems  necessary  to  make  the  specific 
immune  body  really  active. 

The  Side  Chain  Theory.  Thus  far  we  have  been  concerned  with 
the  results  of  experiments,  with  facts  about  which  there  cannot  be 
much  question.  But  a  comprehensive  theory  to  correlate  all  these  gen- 
eralizations became  necessary.  Many  attempts  have  indeed  been  made 
to  establish  a  theoretical  basis  for  the  doctrines  of  immunity,  but  it 
remained  for  Ehrlich  to  suggest  something  which  is  really  tangible 
from  the  chemical  standpoint.  To  follow  the  Ehrlich  notions  some 
other  matters  must  be  explained  first. 

Years  ago,  in  attempting  to  explain  some  of  the  properties  of  large 
organic  molecules,  Pasteur  introduced  the  notion  of  molecular  asym- 
metry into  chemical  science.  He  showed  the  value  of  the  notion  of 
configuration  in  dealing  with  certain  classes  of  chemical  problems. 
This  general  idea  was  greatly  advanced  by  Emil  Fischer  in  his  papers 
on  the  chemistry  of  the  sugars.  Certain  ferments  were  found  to 
decompose  one  sugar  of  an  isomeric  group,  but  to  leave  the  other 
almost  identical  sugars  untouched.  In  other  words  the  ferments  were 
found  to  observe  a  specific  selection,  and  to  work  as  ferments,  the 
enzymes  in  question  must  possess  a  certain  stereochemical  structure 
bearing  some  relation  to  the  stereochemical  structure  of  the  sugar. 
Without  this  relation  fermentation  cannot  take  place.  In  order  to 
make  his  meaning  plain  Fischer  employed  a  figure  which  has  since 
become  famous.  He  said,  in  speaking  of  certain  glucosides,  "  Enzyme 
and  glucoside  must  fit  into  each  other  as  a  key  into  a  lock  in  order  that 
the  one  may  be  able  to  exert  a  chemical  action  on  the  other."  In  one 
16 


226  PHYSIOLOGICAL    CHEMISTRY. 

of  his  papers  Fischer  suggested  that  the  idea  of  related  molecular  con- 
figuration of  ferment  and  fermentable  body  may  prove  of  value  in 
physiological  investigations  as  well  as  in  chemistry,  and  in  the  develop- 
ment of  the  theory  of  Ehrlich  this  prediction  has  been  verified.  Toxins 
and  anti  bodies  combine  with  each  other  only  when  they  possess  corre- 
sponding atom  groups,  and  specificity  is  regarded  as  dependent  on  this 
relative  configuration. 

Without  going  into  minute  details  the  chemical  part  of  the  Ehrlich 
theory  is  briefly  this :  Bacteria,  animal  cells  and  toxins  are  all  complex 
aggregations  of  more  or  less  complex  molecules.  The  latter  have  cer- 
tain configurations  dependent  on  the  presence  of  side  chains  or  side 
groups,  to  borrow  an  expression  from  organic  chemistry.  These  side 
chains  are  directly  or  indirectly  the  points  of  attack  or  defense  in  the 
action  of  the  several  bodies  on  each  other.  In  order  that  a  substance 
may  act  as  an  enzymic  poison  or  toxin  to  cells  of  the  body  both  cells 
and  toxins  must  therefore  possess  certain  reciprocal  configurations.  It 
has  been  suggested  further  that  these  side  groups  are  concerned  in  all 
the  actions  of  the  cells  and  that  it  is  through  them,  for  example,  that 
the  latter  absorb  their  necessary  nutriment  and  elaborate  new  struc- 
tures from  it.  Some  of  the  side  chains  may  be  constructed  to  combine 
with  fats,  some  with  carbohydrates  and  some  with  proteins,  but  in  the 
presence  of  toxins  or  bacteria  with  the  right  kind  of  side  chains  com- 
bination with  these  may  take  place  instead.  Many  of  these  combina- 
tions, perhaps  all,  take  place  not  directly,  but  indirectly,  through  the 
presence  of  an  intermediary  body  or  group  which  itself  must  possess 
two  linking  complexes  or  groups  with  proper  configurations. 

To  describe  these  various  groups  certain  special  names  have  been 
suggested.  Immune  body  is  the  specific  substance  formed  in  the  im- 
munizing process  against  cells  and  is  known  also  by  several  other 
names,  among  which  amboceptor  and  intermediary  body  are  the  most 
commonly  used.  The  complement,  addiment  or  alexin  is  the  ferment- 
like body  found  in  normal  fresh  serum,  and  which  added  to  the  immune 
body  makes  up  the  real  cytotoxin.  It  is  not  specific  and,  as  intimated 
above,  is  sensitive  to  heat,  and  also  to  light  and  air  (oxygen).  The 
various  groups  of  the  large  cell  complex,  whether  of  a  body  cell  or  of 
a  bacterium,  which  have  the  power  of  uniting  with  other  groups  are 
called  receptors.  The  part  of  the  receptor  which  is  free  to  combine 
with  a  food  molecule  or  analogous  substance  is  called  its  haptophorous 
group.  Ehrlich  pictures  the  part  played  by  the  immune  body  and  the 
complement  in  this  way.  Assuming  that  a  bacterial  cell  enters  a  me- 
dium where  these  complexes  are  present  the  immune  body  attaches 


SPECIAL  PROPERTIES  OF  BLOOD  SERUM.  227 

itself  to  the  haptophorous  group  of  the  cell.  In  this  condition  no 
action  follows  immediately;  but  the  immune  complex  has  itself  two 
haptophorous  groups  or  side  chains,  through  one  of  which  the  union 
with  the  bacterial  cell  is  effected,  while  with  the  other  it  joins  on  to  the 
addiment  or  complement  through  its  haptophorous  group.  In  this  way 
the  complement,  which  alone  is  inactive  or  unable  to  attack  the  cell,  is 
brought  into  the  immediate  neighborhood  of  the  latter,  where  its  pro- 
teolytic efforts  are  more  effective.  Every  fresh  normal  serum  seems 
to  have  present  enough  of  the  complement  groups;  the  question  of 
destroying  the  invading  cell  depends  then  on  the  number  of  immune  or 
intermediary  groups  in  the  field.  Another  part  of  the  Ehrlich  theory 
attempts  to  account  for  these. 

The  Immune  Group.  Ehrlich  traces  the  development  of  the  im- 
mune body  to  the  spontaneous  effort  on  the  part  of  the  cell  to  protect 
and  regenerate  itself  in  case  of  partial  destruction.  The  various  cells 
of  the  body  exist  in  a  kind  of  equilibrium  with  each  other.  An  injury 
to  one,  that  is  the  loss  of  some  of  its  side  chains,  immediately  leads  to 
an  effort  at  compensation.  The  hyperplasia  observed  in  an  organ  may 
extend  to  the  single  cells  and  in  consequence  of  this  we  have  over- 
compensation. The  cell's  efforts  at  regeneration  lead  to  the  produc- 
tion of  more  side  chains  than  are  actually  necessary  and  some  of  these 
combine  with  the  aid  of  their  receptor  groups  with  toxin  or  with  com- 
plement. Many  are  formed  in  excess  and  are  thrown  off  into  the  circu- 
lation. These  free  receptors  constitute  the  various  anti  or  immune 
bodies.  Combining  with  complement  groups  they  form  the  true  cyto- 
toxins.  The  larger  the  number  of  free  receptors  thrown  off  into  the 
blood  by  the  over-compensating  efforts  of  the  attacked  cells  the  stronger 
is  its  cytotoxic  or  antitoxic  character  since  these  receptors  hold  either 
the  toxin  or  foreign  cell  and  thus  protect  the  parent  native  cell  from 
attack  or  destruction. 

The  fundamental  point  then  in  the  Ehrlich  theory  of  serum  immunity 
is  this  formation  of  side  chains  in  excess  by  over-compensation,  and  is 
founded  on  the  somewhat  earlier  Weigert  doctrine  of  cell  regeneration 
and  over-compensation  in  general.  Ehrlich  has  added  the  chemical 
conceptions  of  side  chain  groups  and  has  drawn  numerous  illustrations 
from  organic  chemistry  to  show  how  they  may  act.  Large  molecules 
holding  amino,  sulphonic  acid  or  halogen  addition  groups,  for  example, 
may  lose  these  or  take  them  on  again  or  take  others  like  them  without 
losing  their  identity.  Reagents  acting  on  such  a  large  molecule  attack, 
not  the  nucleus,  but  these  side  chains  in  general.  The  simple  organic 
molecule  has  not  the  power  of  self  regeneration,  but  the  cell,  which  is 


228  PHYSIOLOGICAL    CHEMISTRY. 

a  collection  of  many  such  molecules,  has  the  power  of  forming  new 
materials  from  the  nutritive  substances  furnished  to  it.  If  whole  new 
cells  are  formed  why  not  parts  of  cells  or  the  outlying  side  groups  as 
well,  and  this  is  the  Ehrlich  assumption,  which  is  not  unreasonable. 

As  explained  above  these  side  chains  or  receptors  are  of  various 
kinds.  Three  distinct  types  or  orders  are  easily  recognized.  Recep- 
tors of  the  First  Order  have  one  haptophorous  group  and  form  anti- 
toxins. That  is,  they  combine  chemically  with  the  soluble  toxins  in  the 
serum  and  in  a  sense  neutralize  them.  Receptors  of  the  Second  Order 
have  one  haptophorous  group  with  which  a  foreign  molecule  or  group 
may  be  held  and  one  special  group  which  performs  the  function  of  an 
agglutinin  or  precipitin.  Receptors  of  the  Third  Order  or  amboceptors 
have  two  haptophorous  groups  with  which  two  things  may  be  united. 
One  of  these  is  the  foreign  cell  (through  its  corresponding  hapto- 
phorous group)  and  the  other  the  complement.  In  this  way  the  com- 
plement or  alexin  is  able  to  work  on  the  invading  cell  and  attack  it 
through  its  "  zymotoxic  "  group.  These  amboceptors  are  in  themselves 
inactive  and  can  behave  as  cytotoxins  (hemolysins  or  bacteriolysins) 
only  when  joined  to  the  complement  or  ferment  group.  They  are 
formed  in  the  serum  by  immunization  with  foreign  cells,  and  in  turn 
combine  with  cells. 

Another  product  of  immunization  with  cells  is  the  agglutinin  recep- 
tor, while  immunization  with  toxins  leads  to  the  formation  of  receptors 
of  the  First  Order.  Cytotoxins  produced  by  one  animal  species  A, 
brought  into  the  serum  of  another  animal  species  B,  lead  to  the  forma- 
tion of  anticytotoxins  which  may  be  either  anti  complements  or  anti 
amboceptors. 

Toxin  molecules  on  standing  or  by  heating  seem  to  lose  some  of 
their  activity  or  toxic  power,  while  their  power  of  combining  chem- 
ically with  or  neutralizing  antitoxins  is  not  diminished.  In  this  con- 
dition they  are  called  by  Ehrlich  toxoids,  and  he  explains  the  behavior 
by  assuming  that  the  toxins  have  two  characteristic  groups,  one  of 
which,  a  haptophorous  group,  persists  and  combines  with  the  antitoxin 
while  the  other  is  less  stable  and  may  be  lost;  this  he  calls  the  toxo- 
phorous  group.  By  warming  to  55°-6o°  the  complement  bodies  of 
serum  become  converted  into  active  complementoids,  which  retain  the 
haptophorous  group  but  lose  the  zymotoxic  group.  Amboceptors  may 
lose  in  the  same  way  one  of  their  combining  groups  and  become 
amboceptoids. 

In  the  further  development  of  the  Ehrlich  nomenclature  the  term 
toxon  was  introduced  to  describe  another  form  of  modified  toxin. 


SPECIAL  PROPERTIES  OF  BLOOD  SERUM.  229 

The  toxons  are  bodies  of  relatively  slight  toxicity,  and  exist  with  the 
toxin  from  the  start,  in  place  of  being  developed  on  standing.  They 
have  the  power  of  combining  with  antitoxins. 

It  would  not  be  proper  in  this  place  to  go  more  fully  into  the  details 
of  the  Ehrlich  theory ;  enough  has  been  given  to  furnish  the  student  an 
outline  of  the  most  important  points  in  the  theory.  It  is  of  course  true 
that  much  of  the  present  view  is  artificial  and  tentative  and,  with  closer 
fixation  of  facts,  must  be  modified.  This  has  been  the  history  of  the 
development  of  all  chemical  theories.  In  its  main  features  the  Ehrlich 
doctrine  gives  us  a  tangible  picture  of  how  the  serum  may  act  toward 
foreign  bodies.  For  the  ultimate  reasons  for  the  formation  of  immune 
side  chains  by  stimulation  we  have  no  more  explanation  than  we  have 
for  many  of  the  manifestations  of  chemical  affinity.  In  its  outlines 
this  theory  of  the  action  of  immune  serum  appears  wholly  fanciful,  but 
in  reality  it  makes  no  greater  claim  on  the  imagination  than  do  some  of 
the  oldest  accepted  theories  of  general  chemistry. 


CHAPTER   XV. 

TRANSUDATIONS  RELATED  TO  THE  BLOOD. 

THE    LYMPH. 

The  capillary  vessels  convey  the  arterial  blood  with  its  store  of  nutri- 
ment to  the  various  tissues,  which  by  transudation  receive  the  required 
amount  of  nourishing  matter.  The  communication  between  the  blood 
vessels  and  the  tissue  cells  is  not  a  direct  one;  on  the  contrary,  this 
transudation  takes  place  into  the  multitudes  of  star-shaped  spaces 
which  break  the  continuity  of  the  cells,  and  which  communicate  with 
each  other  by  means  of  fine  canals.  A  liquid  passes  from  the  blood 
into  this  network  of  spaces  which  form  the  beginning  of  a  new  vascular 
system.  The  transuded  liquid  is  the  lymph  which  serves  the  double 
purpose  of  nourishing  the  tissues  and  draining  them  also,  since  this 
liquid  not  only  gives  up  large  molecules  of  absorbable  matter,  but  takes 
up  at  the  same  time  various  products  of  metabolism.  What  is  left 
over  after  this  contact  with  the  tissues  collects  in  the  minute  lymph 
capillaries  and  then  into  the  larger  lymphatic  circulation  proper. 

Composition.  Being  thus  related  to  the  blood  the  lymph  must  have 
a  composition  not  greatly  different  from  that  of  the  plasma.  The  nor- 
mal lymph  is  a  nearly  clear  fluid  with  a  specific  gravity  somewhat  less 
than  that  of  the  serum  as  a  rule.  It  contains  salts  and  organic  sub- 
stances as  does  the  serum  of  the  blood,  but  is  naturally  poorer  in  protein 
elements  since  a  portion  of  these  has  been  taken  up  to  nourish  the  neigh- 
boring cells.  The  lymph  contains  a  small  amount  of  fibrinogen.  Very 
few  red  corpuscles  are  present,  but  as  the  formation  of  leucocytes  or 
white  corpuscles  takes  place  in  the  so-called  lymphatic  glands  these 
form  elements  are  abundant  in  the  final  flow. 

It  has  been  already  shown  that  potassium  salts  are  common  to  the 
corpuscles  of  the  blood,  while  the  salts  of  sodium  are  abundant  in  the 
plasma.  We  naturally  find  the  same  thing  in  the  lymph  which  con- 
tains, in  addition  to  proteins,  fat,  sugar,  cholesterol,  etc.,  inorganic 
salts,  having  about  the  following  composition,  according  to  some  old 
analyses  by  Schmidt : 

Sodium  chloride    5.67  per  1000 

Sodium  oxide   1.27 

Potassium  oxide    0.16 

230 


TRANSUDATIONS  RELATED  TO  THE  BLOOD.  23 1 

Sulphuric  acid,  S03    0.09  per  1000 

P:05  as  combined  with  alkalies   0.02 

Calcium  and  magnesium  phosphates   0.26 

This  result  is  approximately  what  one  would  expect  from  an  analysis 
of  blood  serum  and  is  in  fact  about  what  has  been  found.  The  two 
fluids  have  the  same  osmotic  pressure,  due  largely  in  both  cases  to 
salt  content. 

Function  of  the  Lymph.  The  amount  of  metabolic  substances 
returned  finally  to  the  venous  circulation  through  the  lymphatics  does 
not  appear  to  be  great.  The  chief  product  of  oxidation,  COo,  seems 
to  be  thrown  back  directly  from  the  lymph  spaces  to  the  smaller  vessels 
leading  to  the  venous  system.  The  lymph  spaces  into  which  the  trans- 
uded serum  flows  have  apparently  two  ways  of  discharge.  The  bulk 
of  the  liquid  with  some  of  the  absorbed  metabolic  products  passes,  as 
intimated,  into  the  gradually  enlarging  lymphatic  system,  but  certain 
other  complexes,  and  among  them  possibly  the  most  abundant  oxida- 
tion products,  evidently  find  their  way  immediately  into  the  capillary 
beginnings  of  the  venous  circulation.  It  is  not  possible  to  give  exact 
figures  as  to  the  relative  amounts  of  these  products  going  the  two 
courses,  but  it  is  accurately  known  that  the  carbon  dioxide  pressure  in 
the  lymph  is  much  less  than  in  the  venous  circulation,  and  the  urea 
appears  also  to  be  less. 

It  appears  to  be  pretty  well  established  that  the  leucocytes  are  active 
in  hastening  the  destruction  of  complex  products  of  tissue  waste. 
These  cells  seem  to  possess  a  marked  chemical  activity  which  is  mani- 
fested in  a  kind  of  digestion  of  the  grosser  complexes  separated  in  the 
tissue  metabolism,  but,  unfortunately,  our  knowledge  here  is  meager. 
The  normal  end  products  of  this  breaking  down  process  are  not  formed 
at  once.  Possibly  the  leucocyte  is  one  of  the  assisting  agents.  It  has 
been  therefore  held  by  many  writers  that  the  formation  of  these  lymph 
cells  is  probably  the  most  important  part  of  the  work  in  the  lymphatic 
system. 

The  amount  of  lymph  which  is  formed  daily  is  relatively  large  and 
possibly  equal  to  the  volume  of  blood.  A  large  part  of  this  comes 
from  the  flow  through  the  lacteals.  Certain  substances  have  the  power 
of  stimulating  the  flow  of  lymph.  Various  salts,  sugars  and  urea  have 
this  property;  they  are  called  lymphagogues.  Muscular  exertion  also 
increases  the  production  of  lymph,  because  it  is  needed  to  supply  tissue 
waste,  and  a  more  rapid  flow  is  called  for  to  carry  off  the  products  of 
disintegration.  Of  the  manner  in  which  the  lymph  gives  up  its  content 
of  nutrients  to  the  tissues  absolutely  nothing  is  known;  but  the  object 


232  PHYSIOLOGICAL    CHEMISTRY. 

of  this  intermediary  system  is  readily  seen.  It  serves  as  a  regulating 
mechanism  to  prevent  too  rapid  changes  in  the  blood  composition  which 
would  follow  if  it  should  come  in  direct  contact  with  the  tissues. 

CHYLE. 

During  the  digestion  of  fatty  foods  the  lymph  absorbed  from  the 
intestinal  walls  contains  numerous  minute  fat  globules  in  the  form  of 
an  emulsion.  This  portion  of  the  lymph  is  known  as  chyle  and  is 
carried  along  by  the  lacteals  and  finally  discharged  into  the  lower  part 
of  the  thoracic  duct. 

In  composition  chyle  differs  from  the  lymph  from  other  sources 
mainly  in  its  fat  content.  In  the  periods  when  digestion  is  not  in 
progress  the  lacteal  lymph  is  also  clear.  These  vessels  are  then  partly 
collapsed  and  hard  to  see. 

TRANSUDATIONS   PROPER. 

The  lymph  has  sometimes  been  considered  a  transudation  of  the 
blood,  but  the  term  is  now  more  commonly  used  to  describe  the  flow 
of  liquid  from  the  blood  into  certain  cavities  of  the  body  under  patho- 
logical conditions.  A  transudation  proper  is  then  a  modified  lymph 
and  results  often  from  an  imperfect  elimination  of  water  by  the  kid- 
neys, or  from  some  disturbance  in  the  circulation.  Inflammatory 
transudations  are  sometimes  distinguished  as  exudations,  and  in  these 
the  cell  elements  are  much  increased.  If  they  are  excessive  the  dis- 
charge is  known  as  pus. 

For  example,  the  pleural  and  peritoneal  cavities  contain  but  little 
fluid.  The  serous  surfaces  are  moist,  but  it  would  not  be  possible  to 
collect  enough  fluid  substances  for  satisfactory  analysis  under  normal 
conditions.  In  the  advanced  stage  of  pleurisy  a  considerable  quantity 
of  fluid  collects  in  the  pleural  cavity  and  its  composition  resembles  that 
of  the  lymph,  but  it  is  poorer  in  solids  ordinarily.  In  some  forms  of 
acute  peritonitis  a  collection  of  similar  fluid  may  take  place  in  the  peri- 
toneal cavity  and  this  may  amount  in  bad  cases  to  several  liters. 

The  various  forms  of  dropsy  described  by  physicians  are  essentially 
characterized  by  analogous  transudations  of  serous  fluid  without  in- 
flammation. Ascites,  or  dropsy  of  the  abdomen,  hydrocele,  or  dropsy 
of  the  testicle,  and  hydrothorax,  dropsy  of  the  pleura,  are  illustrations. 
Some  analyses  are  given  below,  showing  the  general  nature  of  the  fluids 
collected  in  such  cases.  It  must  not  be  supposed,  however,  that  exactly 
similar  results  would  always  be  obtained  by  analyses  of  fluids  from  the 


TRANSUDATIONS    RELATED    TO    THE   BLOOD.  233 

same  organs.     The  composition  of  pus  serum  is  somewhat  similar;  it 
contains,  however,  more  products  of  protein  disintegration. 

Hydrocele 

Fluid  Pus  Serum 

(Hammarsten).  (Hoppe-Seyler). 

Water  938.85  Water  909.63 

Serum  albumin   35-94  Proteins   70.22 

Globulin     13.25  Lecithin    1.03 

Fibrin   O.59  Fat   O.27 

Ether  extractives    4.02  Cholesterol 0.70 

Soluble   salts    8.60  Alcohol  extractives    1.13 

Insoluble  salts 0.66  Water  extractives  9.22 

Salts    7.75 

Pleural  Peritoneal 

Transudate  Transudate 

(Scherer).  (Hoppe-Seyler). 

Water  935-52  Water  969.64 

Albumin  49-77  Albumin  19.29 

Fibrin    ." 0.62  Urea    0.31 

Ether  extract  2.14  Ether  extract  0.43 

Alcohol  extract  1.84  Alcohol  extract  1.37 

Water  extract   1.62  Water  extract    0.98 

Salts    7.93  Salts    7.98 

Amniotic  Fluid.  This  may  be  considered  as  a  kind  of  transudate. 
A  number  of  analyses  have  been  made  which  show  about  98.5  per  cent 
of  water,  1  per  cent  of  salts  and  0.5  per  cent  of  organic  solids,  largely 
proteins. 

THE   LYMPH    CELLS. 

These  large  cells  or  leucocytes  have  already  been  referred  to  as 
formed  in  the  lymph  glands.  They  are  also  formed  in  large  numbers 
in  the  spleen  and  the  thymus  gland.  From  whatever  source  produced 
they  are  supposed  to  have  the  same  general  composition  and  chemical 
function.  The  following  analysis  by  Lilienfeld  gives  an  idea  of  their 
general  composition.  The  dry  substance  of  the  cells  amounted  to  1 14.9 
parts  per  1000  and  was  made  up  of  the  following  constituents,  the 
figures  referring  to  per  cent  amounts  of  the  dry  matter.  The  cells 
analyzed  were  from  the  thymus  of  the  calf. 

Leuco-nuclein    68.79 

Albumins    1.76 

Histone  8.67 

Lecithin    7.51 

Fat    4.02 

Cholesterol   4.40 

( i!;,<  ogen  0.80 


2  34  PHYSIOLOGICAL    CHEMISTRY. 

In  addition  to  these  organic  substances  mineral  matters  are  present, 
with  salts  of  potassium  characteristic.  The  substance  described  as 
leuco-nuclein  is  apparently  the  nucleic  acid  complex  which  in  the  orig- 
inal cell  is  combined  with  the  histone  as  a  nucleate.  The  lecithin  is  an 
important  fraction  in  these  cells,  and  may  exist  in  part  as  a  protein 
combination.  The  methods  of  separating  lecithin  are  far  from  exact. 
Pus  Cells.  In  their  origin  and  characteristics  these  may  be  consid- 
ered as  very  similar  to  the  leucocytes,  if  not  indeed  identical  with  them. 
The  few  analyses  made  show  a  general  agreement  when  reduced  to 
the  same  terms.  It  must  be  remembered  that  such  analyses  are  far 
from  simple  operations,  especially  in  the  separation  of  the  several  pro- 
tein constituents.  The  following  figures  by  Hoppe-Seyler  should  be 
compared  in  that  light  with  the  above.  The  numbers  refer  as  before 
to  the  dry  matter : 

Nuclein  and  albumin   67.40 

Lecithin 7.56 

Fat   7.50 

Cholesterol    7.28 

Cerebrin  and  extractives    10.28 

Cerebrin  is  the  name  given  to  a  body  containing  nitrogen  in  small 
amount,  but  which  is  not  a  protein.  It  is  usually  found  in  products 
derived  from  the  brain.     As  to  its  exact  nature  but  little  is  known. 

The  pus  cells  float  in  a  fluid  known  as  the  pus  serum,  which  closely 
resembles  the  other  transudates,  as  shown  by  the  analysis  above.  The 
cells  may  be  separated  from  the  serum  by  the  centrifuge,  and  if  mixed 
with  a  little  strong  alkali  yield  a  gelatinous  slime,  which  is  character- 
istic. This  test  is  sometimes  applied  for  the  detection  of  pus  in  urine. 
On  bacterial  decomposition  pus  yields  a  number  of  products  easily  rec- 
ognized as  derived  from  the  nuclein  fraction  of  the  protein. 

The  Spleen.  While  our  knowledge  of  the  functions  of  the  spleen 
is  very  imperfect,  a  few  words  may  be  said  in  this  connection,  since  as 
far  as  is  known  the  lymph  glands  in  producing  leucocytes  do  about  the 
same  kind  of  work.  The  one  thing  most  apparent  about  the  spleen  is 
that  in  this  organ  large  numbers  of  white  cells  are  formed  and  given 
to  the  blood ;  it  is  also  known  that  these  cells  suffer  destruction  there, 
as  the  spleen  pulp  contains  considerable  quantities  of  the  xanthine 
bases,  which  are  among  the  common  products  of  cell  nuclei  destruction. 
The  cells  so  destroyed  may  possibly  be  those  which  have  already  served 
their  purpose  in  the  blood  as  the  disintegrating  agents  concerned  in  the 
breaking  down  of  other  bodies.     Uric  acid,  as  derived  from  the  xan- 


TRANSUDATIONS    RELATED    TO   THE   BLOOD.  235 

thine  bases,  is  known  to  result  when  blood  is  rubbed  up  with  the  spleen 
substance.  The  spleen  is  enlarged  in  many  cases  of  infectious  diseases. 
This  is  possibly  from  the  abnormally  great  production  of  leucocytes 
needed  in  the  blood  in  overcoming  the  effects  of  the  toxic  agents  or 
invading  bacteria. 

Of  the  chemical  nature  of  the  spleen  substance  little  is  known,  as  it 
is  practically  impossible  to  free  it  from  blood  for  analysis.  In  addition 
to  the  xanthine  bodies  and  other  decomposition  products  there  seems 
to  be  present  an  albuminous  substance  containing  iron,  which  is  con- 
sidered as  an  albuminate;  but  of  its  uses  nothing  definite  is  known, 
beyond  the  possibility  that  it  may  be  concerned  in  the  production  of  red 
corpuscles. 

The  chemical  work  performed  by  the  spleen  may  evidently  be  done 
by  other  organs,  as  it  may  be  completely  extirpated  without  leading  to 
fatal  results.  In  its  absence  the  production  of  great  numbers  of  leuco- 
cytes falls  to  the  lymph  glands,  and  the  red  marrow  of  bones  may  take 
over  the  work  of  generating  red  blood  corpuscles. 


CHAPTER   XVI. 

MILK. 

The  qualitative  composition  of  milk  as  produced  by  the  mammary- 
glands  of  different  animals  is  nearly  the  same  whatever  the  species  of 
animal.  But  in  quantitative  composition  very  great  differences  obtain. 
Cow's  milk  has  always  been  taken  as  the  type  with  which  comparisons 
are  made,  as  it  is  the  kind  everywhere  in  general  use.  The  essential 
differences  between  it  and  mother's  milk  will  be  pointed  out  in  what 
follows. 

COW'S   MILK. 

In  an  earlier  chapter  an  analysis  of  cow's  milk  is  given  which  repre- 
sents a  general  average  of  composition  of  good  market  milk.  But  the 
normal  milk  of  individual  cows  may  be  very  different  from  that  there 
described.  The  qualitative  composition  is  always  the  same,  but  in  the 
amounts  of  fat,  sugar  and  protein  present  the  greatest  divergences  are 
noticed.  These  variations  depend  on  the  race  of  the  animal,  the  period 
of  lactation  and  especially  on  the  feed.  It  is  also  a  well-known  fact 
that  the  richness  of  milk  varies  during  the  time  of  milking,  the  first 
portions  of  milk  withdrawn  from  the  udder  being  poorer  in  fat  than 
the  last  part  or  "  strippings."  In  speaking  of  normal  milk,  then,  these 
facts  must  be  kept  in  mind ;  a  milk  may  be  normal  but  not  necessarily 
rich  or  good,  from  the  standpoint  of  food  value.  The  following  table 
illustrates  the  variations  found  in  the  analyses  of  milk  from  a  large 
number  of  cows.     The  mean  specific  gravity  is  from  1.029  to  1.033. 

Mean.  Maximum.  Minimum. 

Water    87.4  91.5  84.0 

Fat    3.5  6.2  2.0 

Sugar 4.5  6.1  2.0 

Proteins    3.9  6.6  2.0 

Salts    0.7  1.0  0.3 

When  the  water  of  milk  is  found  as  high  as  91.5  per  cent  of  the 
whole  the  sum  of  the  fat,  protein,  sugar  and  salts  can  be  only  8.5  per 
cent  in  place  of  12.5  per  cent,  which  should  be  expected  in  the  mixed 
market  milk. 

Market  Milk.  Experience  has  shown  that  the  mixed  milk  from  a 
herd  of  well-kept  cows  should  have  a  composition  not  far  from  that 

236 


MILK.  237 

given  in  the  table  above  under  "mean."  Laws  have  been  passed  in 
most  of  the  large  cities  of  the  United  States  and  Europe  requiring  that 
milk  sold  as  pure  must  be  of  a  quality  not  inferior  to  this  mean  value. 
Indeed,  in  some  places  a  market  milk  of  still  higher  standard  is  required. 

PHYSICAL  COMPOSITION  OF  MILK. 

The  exact  nature  of  the  mixture  of  the  component  parts  of  milk  has 
long  been  a  debated  question.  When  taken  from  the  udder  the  fats, 
proteins,  sugar  and  salts  are  mixed  homogeneously,  and  no  immediate 
tendency  is  observed  toward  a  separation  of  the  light  fat  from  the 
other  and  heavier  solids.  In  time,  however,  such  a  separation  takes 
place  and  the  fat  rises  in  the  form  of  cream.  Milk  cannot  be  looked 
upon  as  a  transudation  from  the  blood  because  it  contains  substances 
not  found  in  that  fluid;  the  casein  of  milk  and  the  lactose  are  different 
from  the  proteins  and  sugar  normally  existent  in  the  blood,  and  the 
fat  of  milk  is  more  complex  probably  than  the  blood  fat.  It  is  neces- 
sary to  admit,  then,  that  some  of  the  milk  components  are  produced  in 
or  from  the  substance  of  the  mammary  glands  themselves.  It  is  held 
by  some  authorities  that  the  nucleo-proteids  of  the  gland  cells  are 
similar  to  or  identical  with  the  casein,  which  therefore  has  its  origin 
in  the  gradual  breaking  down  of  those  cells.  In  regard  to  the  milk 
fat  it  is  known  that  certain  fats  can  pass  but  little  changed  from  the 
food  through  the  blood  and  appear  finally  in  the  milk,  imparting  pecu- 
liar properties.  But,  on  the  other  hand,  milk  fat  is  produced  when  the 
food  of  the  animal  contains  no  fat  whatever,  and  certainly  no  fats 
resembling  the  characteristic  volatile  fats  of  the  milk.  In  the  car- 
nivora,  confined  to  an  essentially  protein  diet,  milk  fat  is  formed,  and 
in  the  herbivora  on  a  diet  containing  largely  pentoses  and  other  carbo- 
hydrates milk  fat  is  likewise  produced  normally  and  in  quantity. 

These  facts,  then,  seem  to  be  clear,  that  while  under  some  circum- 
stances fats  as  such  pass  from  the  blood  into  the  milk,  and  this  is  further 
evident  by  the  experience  of  feeding  cows  with  certain  foods  rieh  in 
fats,  the  milk  glands  have  the  power  of  producing  the  several  individual 
fats  as  occurring  in  milk  from  compounds  which  are  not  fat  to  begin 
with.  In  discussing  the  chemistry  of  proteins  in  an  earlier  chapter  it 
was  shown  that  in  the  breaking  down  of  these  bodies  under  the  influ- 
ence of  various  agents  fatty  acids  are  found  among  the  decomposition 
products.  The  complex  protein  molecule  may  be  all  that  is  necessary 
to  give  rise  to  the  milk  fats  if  other  things  are  not  available. 

The  origin  of  milk  sugar  is  not  at  all  clear.  Lactose  is  not  a  con- 
stituent of  our  ordinary  foods  and  at  best  the  blood  contains  probably 


238  PHYSIOLOGICAL    CHEMISTRY. 

only  inverted  sugars  or  monosaccharides.  In  some  cases  the  forma- 
tion of  milk  sugar  may  be  traced  indirectly  to>  the  carbohydrates  of  the 
food ;  but  this  will  not  explain  the  production  of  sugar  in  the  carnivora. 
Here  as  before  we  are  probably  obliged  to  fall  back  on  the  behavior  of 
the  complex  proteins.  Among  the  groups  they  contain,  or  at  any  rate 
yield  in  decomposition,  the  presence  of  sugar  groups  has  been  certainly 
shown.  This  was  explained  in  a  former  chapter.  The  milk  lactose 
probably  results  from  a  synthesis  of  these  simpler  sugars. 

From  what  is  in  general  known  of  the  nature  of  complex  protein 
matter  such  as  exists  in  the  milk  glands  it  seems  therefore  possible  to 
trace  the  origin  of  the  milk  proteins,  sugar  and  fats  to  the  disintegra- 
tion of  this  original  protein  substance.  But  of  the  agents  of  disin- 
tegration, and  following  necessary  syntheses,  we  know  absolutely 
nothing.  The  presence  of  certain  enzymes  has  been  assumed,  but  as 
they  have  not  been  isolated  or  identified,  their  part  in  the  reactions 
remains  speculative. 

CHEMISTRY  OF  THE  MILK  COMPONENTS. 

Fats.  In  the  older  literature  milk  fat  was  given  a  comparatively 
simple  composition.  It  was  assumed  to  consist  of  stearin,  palmitin, 
olein  and  butyrin  essentially,  the  last  named  volatile  fat  imparting  the 
flavor  to  the  separated  butter.  At  the  present  time  we  must  admit 
that  our  knowledge  is  far  from  exact  on  the  subject,  but  we  know 
that  the  composition  of  milk  fat  is  by  no  means  as  simple  as  once 
assumed.  In  the  chapter  on  the  fats  an  analysis  is  given  which  con- 
forms better  to  our  modern  notions.  We  find,  then,  besides  butyrin 
several  glycerol  esters  of  the  same  series  of  comparatively  volatile 
acids.  Among  the  heavier  fatty  acids  myristic  acid  seems  to  have 
some  importance,  as  disclosed  by  a  number  of  analyses.  A  small 
amount  of  lecithin  appears  to  be  also  present. 

Although  produced  from  a  variety  of  materials  in  feeding  experi- 
ments, milk  fat,  as  butter,  maintains  a  rather  constant  composition  as 
disclosed  by  both  chemical  and  physical  tests.  The  melting  point  of 
the  fat  is  usually  between  31  °  and  32. 5  °  C.  and  its  specific  gravity  at 
380  is  about  0.912.  Butter  fat  is  easily  saponified  and  from  the  sapon- 
ified mass  the  fatty  acids  which  are  non-volatile  and  practically  insol- 
uble in  hot  water  may  be  separated.  These  insoluble  acids  amount  in 
the  mean  to  about  87.5  per  cent  of  the  weight  of  the  original  butter 
fat.  That  is,  10  grams  of  average  butter  fat  should  yield  8.75  gm. 
of  insoluble  acids,  the  difference  representing  the  lighter  soluble  fatty 
acids  and  glycerol.     In  the  fat  from  very  rich  milk  the  insoluble  acids 


MILK.  239 

may  be  somewhat  lower  than  this,  while  in  poor  milk  they  would  be 
higher.  These  facts  are  important  in  distinguishing  between  butter 
and  its  substitutes. 

Fat  Globules.  In  milk  the  fat  exists  in  the  form  of  minute  globules 
of  different  sizes.  The  diameters  of  these  globules  vary  between  about 
0.0016  mm.  and  0.01  mm.  A  cubic  centimeter  of  normal  milk  con- 
taining 3.5  per  cent  of  fat  contains  100  millions  or  more  of  these 
globules.  In  the  milk  they  are  described  as  existing  in  the  form  of 
an  emulsion,  but  of  the  exact  nature  of  this  emulsion  our  knowledge  is 
imperfect.  It  has  been  held  also  that  a  membrane  of  casein  encloses 
the  fat  globules  and  that  this  prevents  the  ready  extraction  of  fat 
when  ether  or  similar  solvent  has  been  added  to  milk.  If  the  milk  is 
previously  shaken  with  a  little  acid  which  is  supposed  to  break  or 
destroy  this  membrane  the  ether  added  will  now  dissolve  it.  But  this 
membrane  cannot  be  directly  detected  with  the  microscope,  and  experi- 
ments on  the  formation  of  fat  emulsions  by  the  aid  of  casein  and  weak 
alkalies  have  shown  that  the  presence  of  a  membrane  is  not  necessary 
to  account  for  the  round  form  or  the  failure  to  dissolve  readily  in 
ether.  The  surface  of  the  globule  is  not  the  same  as  the  interior  por- 
tion, as  it  appears  to  take  a  stain  by  certain  agents  which  does  not  pene- 
trate. But  in  the  conflict  of  views  advanced  it  is  not  yet  known  what 
the  surface  actually  is. 

Casein  and  Lactalbumin.  These  compounds  have  been  mentioned 
in  the  chapter  on  proteins  and  their  place  in  the  general  scheme  of 
classification  pointed  out.  In  the  free,  pure  state  the  casein  is  a  dis- 
tinctly acid  body  which  neutralizes  alkali  and  forms  salts  with  rather 
sharply  defined  properties.  Casein  may  be  easily  separated  from  milk 
in  this  way : 

Experiment.  Dilute  500  cc.  of  skimmed  milk  with  about  2  liters  of  water  in  a 
large  jar;  add  enough  dilute  acetic  acid  to  make  not  over  0.1  per  cent  of  the  whole. 
This  causes  a  precipitation  of  the  casein  in  fine  white  flakes  which  soon  settle,  leav- 
ing a  nearly  clear  whey.  After  some  hours  decant  this  whey  and  add  a  greater 
volume  of  distilled  water  and  stir  up  well.  Allow  this  mixture  to  settle  and  pour 
off  the  water.  Add  a  liter  of  water  and  enough  weak  sodium  or  ammonium 
hydroxide  to  dissolve  all  the  casein  and  produce  an  opalescent  solution.  This  in 
turn  is  reprecipitated  with  dilute  acetic  acid  after  adding  considerable  water,  and 
these  operations  are  repeated  several  times.  In  this  way  a  casein  nearly  free  from 
calcium  salts  is  obtained.  It  is  washed  well  with  water  by  decantation,  then  poured 
on  a  Buchner  funnel,  drained,  washed  with  alcohol,  until  the  water  is  removed  and 
finally  several  times  with  ether  to  take  out  the  fat.  On  drying  a  fine  white  powder 
is  obtained   with  which  the  important  properties  of  casein  may  be  shown. 

Experiment.  Weigh  out  5  to  10  gms.  of  casein  into  a  beaker  or  flask  and  add 
distilled  water.  Note  that  it  appears  to  be  quite  insoluble  (This  might  be  shown 
by  filtering  and  testing  the  filtrate  by  evaporation.)     Add  a   few  drops  of  phenol- 


24O  PHYSIOLOGICAL    CHEMISTRY. 

phthalein  reagent  and  run  in  standard  sodium  hydroxide  solution  until  a  permanent 
pink  appears.  In  this  way  the  equivalent  or  combining  weight  of  the  casein  may 
be  found  very  closely.     It  is  over  1000. 

The  alkali  salt  of  the  casein  forms  a  somewhat  viscid  solution.  If  exposed  to 
the  air  it  dries  down  to  a  gummy  mass  which  is  very  adhesive  and  acts  like  a 
mucilage.  In  the  arts  similar,  but  crude,  solutions  are  used  as  sizing  material  and 
as  a  constituent  of  certain  paints.  These  products  are  made  from  the  cheap  whey 
from  the  creameries. 

Experiment.  While  the  alkali  salts  of  casein  are  readily  soluble  in  water  the 
heavier  metal  combinations  are  not.  This  may  be  shown  by  adding  to  the  alkali 
solution  as  obtained  above  solutions  of  salts  of  other  metals.  Precipitates  are  formed 
readily  with  most  of  them.  The  calcium  salt  is  moderately  soluble,  as  may  be  shown 
by  rubbing  up  some  casein  with  calcium  carbonate  and  water.  On  filtering  and 
adding  a  drop  of  acetic  acid  to  the  filtrate  a  casein  precipitate  comes  down. 

Experiment.  The  lactalbumin  may  be  shown  by  boiling  the  decanted  liquid  from 
the  first  acetic  acid  precipitation  given  above.  A  coagulum  forms  as  in  a  dilute 
white  of  egg  solution  to  which  a  little  acid  had  been  added. 

The  phosphorus  in  casein  appears  to  be  combined  in  at  least  two 
forms.  On  digesting  casein  with  pepsin  and  hydrochloric  acid  a 
product  known  as  pseudo-nuclein  is  separated  because  of  its  failure  to 
digest.  In  long  continued  digestion  some  phosphorus  seems  to  pass 
into  the  form  of  orthophosphoric  acid,  while  another  portion  remains 
in  the  albumoses  formed,  in  the  organic  condition.  The  digestion 
residue,  however,  is  not  nucleic  acid,  which  distinguishes  the  casein  from 
the  true  nucleo-proteids.  In  the  precipitation  of  casein  from  milk  by 
the  treatment  given  above  the  combined  phosphorus,  whether  in  acid 
or  organic  combination,  does  not  appear  to  be  touched.  The  mineral 
phosphates  are  separated,  however,  but  not  completely,  as  the  finally 
washed  and  dried  casein  always  contains  a  trace  of  ash,  a  part  of  which 
is  calcium  phosphate.  This  ash  probably  has  nothing  to  do  with  the 
true  casein,  but  is  present  because  of  imperfect  separation. 

Milk  Sugar.  This  crystallizes  with  one  molecule  of  water, 
C12H2201:l  -f-  H20,  and  yields  glucose  and  galactose  on  inversion.  It 
is  separated  in  large  quantities  from  the  whey  of  cheese  factories  and 
is  employed  in  the  manufacture  of  invalid  and  infant  foods.  The 
general  properties  of  the  sugar  have  already  been  given. 

The  Mineral  Substances  in  Milk.  In  the  analyses  quoted  at  the 
beginning  of  the  chapter  the  ash  of  the  milk  is  given  as  about  0.7  per 
cent  in  the  mean.  This  amount  appears  small,  but  still  it  is  of  the 
highest  importance,  as  it  makes  up  between  5  and  6  per  cent  of  the 
total  solids  of  the  milk.  The  composition  of  milk  ash  has  been  the 
subject  of  many  investigations.  While  it  cannot  represent  exactly  the 
condition  of  the  inorganic  substances  in  the  original  milk,  the  agree- 
ment is  an  approximate  one  and  is  probably  near  enough  for  practical 


MILK.  24I 

purposes.  In  obtaining  ash  for  analysis,  sulphur  and  phosphorus  in 
organic  combination  are  thrown  into  oxidized  form  and  combined  as 
salts,  sulphates  and  phosphates,  in  which  form  we  find  them  in  our 
subsequent  tests.  The  following  figures  from  Konig  represent  the 
composition  of  milk  ash  as  the  mean  of  9  analyses : 

Per  Cent. 

K20   24.06 

Na20   6.0s 

CaO  23.17 

MgO    2.63 

Fe203   0.44 

P263    27.98 

S03    126 

CI   I34S 

Accepting  these  figures  as  fairly  accurate,  and  they  agree  pretty  well 
with  the  results  of  all  analysts  who  have  dealt  with  the  question,  1  liter 
of  average  cow's  milk  would  contain  the  following  amounts  of  the 
several  constituents: 

K20  1-74  gm. 

Na20 0.44  gm. 

CaO    167  gm. 

MgO     0.19  gm. 

Fe,03    0.03  gm. 

P,03    2.02  gm. 

S03   0.09  gm. 

CI  0.97  gm. 

7.15  gm. 

Noteworthy  here  are  the  relatively  large  amounts  of  the  phosphates 
of  calcium  and  potassium.  These  salts  represent  all  the  mineral  mat- 
ters needed  in  nourishing  the  body.  As  found  in  the  milk  they  exist 
in  the  combinations  from  which  they  are  most  readily  assimilated. 

The  Colostrum.  This  is  the  milk  secreted  before  and  for  a  few 
days  after  parturition,  and  is  characterized  by  higher  specific  gravity 
and  content  of  solids.  It  contains  a  large  amount  of  coagulable  pro- 
teins and  therefore  thickens  on  boiling.  Some  idea  of  the  general 
composition  is  given  by  the  following  figures  which  represent  the  means 
of  a  number  of  analyses : 

Per  Cent. 

Water   74-05 

Casein   4-66 

Albumin   13-62 

Fat   343 

Sugar    2.66 

Salts    158 

17 


242  PHYSIOLOGICAL    CHEMISTRY. 

Whey  is  the  fluid  left  after  separation  of  the  larger  part  of  the  fat  and  casein 
in  the  cheese  industry  or  by  analogous  coagulation.  The  sugar  and  salts  remain 
practically  unchanged,  while  the  fat  and  casein  are  reduced  to  traces.  The  lact- 
albumin  left  averages  about  0.5  per  cent. 

Buttermilk  differs  from  ordinary  milk  essentially  in  its  lower  content  of  fat.  It 
is  usually  sour  because  of  being  separated  from  ripened  cream,  and  contains  there- 
fore an  appreciable  amount  of  lactic  acid  formed  at  the  expense  of  some  of  the 
sugar. 

Skimmed  milk  is  in  composition  similar  to  buttermilk  but  is  usually  sweet.  In 
the  modern  methods  of  separation  by  centrifugal  machines  the  fat  may  be  reduced 
to  less  than  half  of  one  per  cent;  the  protein  is  also  somewhat  reduced. 

SOME  EXPERIMENTS  WITH  MILK. 

A  few  simple  tests  may  be  made  to  illustrate  the  composition  of  milk. 

The  Test  for  Fat.  Pour  about  20  cc.  of  milk  in  a  porcelain  dish,  add  an  equal 
volume  of  clean,  dry  quartz  sand  and  evaporate,  with  frequent  stirring,  about  an 
hour  on  the  water-bath.  Then  loosen  the  dry  mass  as  well  as  possible  by  means 
of  a  spatula,  or  glass  rod,  and  pour  over  it  25  cc.  of  light  benzine.  Stir  up  well 
and  cover  with  a  sheet  of  paper  and  allow  to  stand  15  minutes.  Then  pour  the 
liquid  through  a  small,  dry  filter  into  a  small,  dry  beaker,  and  place  this  in  hot 
water  to  volatilize  the  benzine. 

A  residue  of  fat  will  be  left.  Do  not  attempt  to  evaporate  the  benzine  over  a 
flame,  or  on  a  water-bath  under  which  a  lamp  is  burning.  Heat  the  water,  then 
extinguish  the  flame  and  immerse  the  vessel  containing  the  benzine  in  the  hot  water. 

The  Test  for  Sugar.  Measure  out  about  10  cc.  of  milk,  and  dilute  it  with  water 
to  make  200  cc.  Add  to  this  5  cc.  of  a  copper  sulphate  solution,  such  as  is  used  in 
making  the  Fehling  solution  (69.3  gm.  per  liter),  and  then  enough  potassium  or 
sodium  hydroxide  solution  to  produce  a  voluminous  precipitate  containing  copper 
with  all  the  proteins  and  fat.  For  this  purpose  about  3.5  cc.  of  a  1  per  cent  sodium 
hydroxide  solution  will  be  required.  Allow  the  precipitate  to  subside,  pour  or  filter 
off  some  of  the  supernatant  liquid,  and  boil  it  with  Fehling's  solution.  The  charac- 
teristic red  precipitate  forms,  showing  presence  of  sugar. 

Protein  Test.  The  presence  of  proteins  in  milk  can  readily  be  shown  as  fol- 
lows :  Mix  equal  volumes  of  milk  and  Millon's  reagent  in  a  test-tube,  and  boil.  The 
bulky  red  precipitate  which  forms  proves  the  presence  of  the  body  in  question. 

Action  of  Rennet  on  Milk.  The  mucous  membrane  of  the  stomachs  of  most 
animals,  and  especially  that  of  the  young  calf,  contains  an  enzyme  known  as  the 
"  milk  curdling  ferment,"  the  "  rennet  ferment,"  or  rennin,  the  nature  of  which  has 
already  been  explained  in  the  chapter  on  the  ferments. 

A  crude  extract  of  the  mucous  membrane  of  the  stomach  from  the  calf  is  com- 
monly called  rennet  and  has  long  been  in  use  for  the  curdling  of  milk  in  the  produc- 
tion of  cheese.  This  curdling  consists  essentially  in  the  coagulation  or  precipitation 
of  the  casein,  which,  it  will  be  recalled,  is  not  readily  thrown  down  by  the  usual 
methods. 

An  active  rennet  can  be  readily  obtained  by  digesting  the  stomach  of  the  calf 
with  glycerol  or  brine.  If  a  brine  extract  is  precipitated  by  alcohol  in  excess  a 
white  powder  separates,  which  when  collected  and  dried,  has  very  active  properties. 
Several  powders  of  this  description  are  now  in  the  market.  Let  the  student  try  the 
following  experiment  with  such  a  product : 

Experiment.  Warm  some  fresh  milk  to  a  temperature  of  380  to  400  C.  in  a  test- 
tube  or  small  beaker,  then  add  about  half  a  gram  of  commercial  "  rennin,"  and  after 
stirring  it  well  keep  for  15  minutes  at  a  temperature  not  above  400.     Then  as  the 


MILK.  243 

milk  cools  it  assumes  the  consistence  of  a  firm  jelly.  It  is  essential  in  this  experi- 
ment that  the  temperature  be  kept  within  the  proper  limits,  as  the  enzyme  is  not 
active  at  low  temperature  and  it  is,  like  others,  destroyed  by  high  temperature.  The 
casein  or  cheese  which  is  obtained  in  this  way  is  not  the  same  as  that  precipitated 
by  acids  as  it  contains  much  calcium  in  combination.  This  form  of  casein  is 
usually  called  para-casein. 

Repeat  the  experiment  by  adding  about  5  drops  of  a  concentrated  sodium  car- 
bonate solution  to  the  milk  and  then  the  rennet.     Coagulation  now  fails  or  is  partial. 

The  Action  of  Pancreatic  Extract  on  Milk.  The  behavior  of  milk  with  extract 
of  pancreas  is  somewhat  complicated  because  of  the  complex  nature  of  the  milk; 
the  sugar,  the  fat,  and  the  protein  bodies  all  suffer  some  change  under  the  influence 
of  the  several  pancreatic  enzymes. 

The  most  interesting  of  these  changes,  however,  is  that  produced  in  the  proteins, 
and  is  commonly  called  peptonization. 

At  the  present  time  the  digestion,  or  peptonization  of  milk,  is  a  very  common 
practice  in  the  preparation  of  food  for  the  sick  room,  and  can  be  illustrated  by  the 
following  experiment : 

Experiment.  Dilute  about  10  cc.  of  milk  with  an  equal  volume  of  water,  and  add 
half  a  gram  of  sodium  bicarbonate.  Next  add  a  few  drops  of  a  liquid  extract 
of  pancreas,  or  a  very  small  amount  (10  to  20  mg.)  of  one  of  the  concentrated 
"  pancreatin  "  powders  on  the  market.  Shake  the  mixture  and  keep  it  at  a  tem- 
perature of  40  degrees  on  the  water-bath  half  an  hour.  At  the  end  of  this  time 
filter  and  apply  the  peptone  test — potassium  hydroxide  and  dilute  copper  sulphate — 
and  observe  the  pink  color.  As  the  action  of  the  pancreatic  extract  is  continued 
the  liquid  resulting  becomes  very  bitter  from  the  formation  of  digestion  products 
other  than  "  peptone."  The  reaction  should  therefore  be  checked  by  cooling  before 
this  very  bitter  stage  is  reached. 

It  will  be  observed  that  these  experiments  illustrate  the  conditions 
in  two  kinds  of  digestion.  The  pancreatic  digestion  of  proteins  in 
milk  is  favored  by  a  neutral  or  slightly  alkaline  reaction.  Alkali  inter- 
feres with  the  rennet  coagulation.  In  the  stomach  the  clotting  of  the 
milk  is  favored  by  the  combined  action  of  the  acid  and  ferment.  In 
the  normal  stomach  coagulation  the  presence  of  calcium  salts  seems  to 
be  essential.  If  milk  be  treated  with  a  small  amount  of  sodium  oxalate 
solution  and  then  rennet,  coagulation  fails.  Calcium  chloride  solution 
added  later,  the  proper  temperature  being  meanwhile  maintained,  brings 
it  about. 

THE  ANALYSIS  OF  MILK. 

The  above  experiments  suggest  some  of  the  steps  in  the  quantitative 
analysis  of  milk,  a  brief  outline  of  which  follows  : 

Water  and  Total  Solids.  Weigh  out  about  5  grams  in  a  small  platinum  dish 
and  evaporate  to  dryness  over  a  water-bath  which  requires  some  hours.  Then 
transfer  the  dish  to  a  hot  air  oven  and  maintain  at  a  temperature  of  1050  through 
half  an  hour.  Cool  the  dish  in  a  desiccator  and  weigh.  The  loss  of  weight  repre- 
sents the  v.  practically  nothing  else  of  consequence  is  volatile. 

Ash  or  Mineral  Matter.  After  weighing  the  dry  residue  or  total  solids  above 
place  the  dish  on  a  triangle  over  a  clear  Bunsen  (lame  and  heat  until  all  the  organic 
matter,  and  finally  the  excess  of  carbon,  i-   driven  off.      The  ash  left  must  be  per- 


244  PHYSIOLOGICAL    CHEMISTRY. 

fectly  white.  Cool  and  weigh  as  before.  There  is  some  slight  loss  of  volatile 
salts  in  this  ignition. 

Fat.  Where  many  analyses  are  made  as  a  routine  operation,  as  in  the  control 
of  market  milk,  fat  is  generally  now  determined,  by  separating  it  from  the  milk  in 
a  centrifugal  machine  and  reading  off  the  volume.  A  definite  quantity  of  milk  is 
measured  out,  mixed  with  a  little  acid  to  facilitate  the  breaking  up  of  the  fat 
globules,  placed  in  a  special  bottle  with  graduated  neck  or  stem  and  rapidly  rotated. 
The  liberated  fat  collects  in  the  stem  and  is  read  off.  With  the  Babcock  machine 
in  common  use  the  method  is  rapid  and  very  accurate 

Fat  is  very  frequently  determined  by  evaporating  milk  mixed  with  broken  glass 
or  quartz  sand  to  dryness  and  extracting  with  a  good  solvent,  preferably  light 
petroleum  benzine  or  perfectly  anhydrous  ether.  A  better  method  is  to  distribute 
about  s  to  io  grams  of  milk  from  a  pipette  over  the  surface  of  a  strip  of  specially 
prepared  absorbent  paper.  This  is  coiled  up  somewhat  loosely,  placed  in  an  air 
oven,  dried  thoroughly  and  then  transferred  to  a  Soxhlet  extraction  apparatus, 
where  it  is  treated  with  the  solvent  by  percolation  through  two  or  three  hours.  The 
solvent  carries  the  fat  down  into  a  small  weighed  flask.  On  evaporation  of  the 
solvent  the  dry  fat  is  left  and  may  be  so  weighed. 

Sugar.  To  determine  the  sugar,  the  fat  and  proteins  must  be  first  separated, 
which  may  be  conveniently  done  by  the  copper  process  as  illustrated  above.  25  cc. 
of  milk  is  diluted  with  water  to  400  cc.  and  10  cc.  of  the  Fehling  copper  solution 
added.  Then  from  a  corresponding  sodium  hydroxide  solution  (containing  10.2 
gm.  to  the  liter)  alkali  is  added  in  amount  just  sufficient  to  throw  down  a  bulky  pre- 
cipitate containing  all  the  proteins  and  fats  with  the  copper.  This  requires  about 
7  cc.  of  the  alkali.  The  mixture  is  diluted  to  500  cc.  and  a  portion  is  filtered  off  for 
tests.  If  the  precipitation  was  properly  made  a  clear  filtrate  is  secured  which  con- 
tains only  a  trace  of  copper,  and  not  enough  to  appreciably  affect  the  accuracy  of 
titration  by  the  Fehling  solution  as  described  in  an  earlier  chapter.  The  proper 
factor  for  lactose  must  be  used  in  the  calculation. 

The  protein  and  fat  may  be  precipitated  by  use  of  a  solution  of  lead  acetate  or 
mercuric  nitrate  without  dilution.  On  filtering  a  clear  filtrate  is  obtained  which  may 
be  tested  by  the  polariscope.  50  cc.  of  milk  should  be  used,  and  after  precipitation 
and  filtration  made  up  to  100  cc.  for  the  polarization  test.  The  details  cannot  be 
given  here. 

The  Proteins.  If  the  sugar,  fat  and  ash  are  accurately  found  the  proteins  may 
be  estimated  by  difference;  that  is  by  subtracting  the  sum  of  these  from  the  total 
solids  found.  But  this  plan  should  not  be  followed  except  as  a  control.  A  direct 
determination  of  casein  may  be  made  in  this  way:  10  cc.  of  milk  is  diluted  to  50 
and  mixed  with  dilute  acetic  acid  to  produce  complete  precipitation.  Something 
less  than  1.5  cc.  of  10  per  cent  acid  will  be  needed  for  this.  The  precipitate  is 
collected  on  a  Gooch  funnel,  washed  with  water,  hot  alcohol  and  finally  enough 
ether  to  remove  all  the  fat.  What  is  left  is  dried  and  weighed  as  casein.  From 
the  first  filtrate  plus  the  wash  water  the  albumin  may  be  precipitated  by  boiling.  The 
coagulum  is  collected  on  a  Gooch  funnel,  washed  with  water  and  alcohol  and  dried 
as  before. 

It  is  also  possible  to  determine  the  total  nitrogen  by  the  Kjeldahl  method,  and 
multiply  this  by  the  factor  6.25  to  obtain  corresponding  total  protein.  This  gives 
a  fairly  good  control. 

MILK   PRESERVATIVES. 

Milk  shippers  and  dealers  often  attempt  to  keep  milk  from  spoiling — turning  sour 
usually — by  the  addition  of  some  anti-ferment  substance.  The  propriety  of  such  an 
addition  has  been  much  discussed.     In  general  the  use  of  food  preservatives  should 


MILK.  245 

be  kept  within  certain  defined  limits,  as  the  consumer  has  the  right  to  know  what 
he  is  using.  The  chemical  substances  employed  in  this  way  possess  different  degrees 
of  activity.  Boric  acid  and  formaldehyde  have  been  most  frequently  added  for 
the  purpose,  but  they  have  been  rather  generally  condemned  for  this  and  other  foods. 
In  the  case  of  milk  it  is  sometimes  a  question  of  the  lesser  evil;  the  trace  of  for- 
maldehyde required  to  effectually  preserve  it  from  acid  fermentation  is  very  small. 
If  no  more  than  this  minimum  is  used  it  is  not  likely  that  the  harm  from  using 
it  would  be  very  great,  if  at  all  noticeable.  The  use  of  such  milk  is  probably  pre- 
ferable to  that  of  the  sour,  unpreserved  milk  often  used  by  children  in  the  poorer 
quarters  of  our  cities. 

In  many  of  our  large  cities  attempts  are  now  made  to  pasteurize 

a  good  portion  of  the  milk  supply.     The  degree  of  safety  afforded  by 

this  operation,  as  carried  out  in  practice,  is,  however,  very  illusory. 

MOTHER'S   MILK. 

We  turn  now  to  a  short  discussion  of  the  chief  points  of  difference 
between  mother's  milk  and  cow's  milk,  which  is  a  subject  of  great 
practical  importance.  Success  in  substituting  cow's  milk  for  mother's 
milk  in  the  feeding  of  small  children  depends  very  largely  on  the 
extent  and  accuracy  of  our  knowledge  here.  It  is  a  singular  fact  that 
we  know  much  less  about  the  chemistry  of  human  milk  than  we  know 
of  other  milks,  and  this  is  in  part  due  to  the  difficulty  in  securing  a 
perfectly  normal  secretion  for  analysis. 

Because  of  the  presence  of  certain  salts  milk  has  a  so-called  ampho- 
teric reaction,  that  is,  it  shows  an  acid  behavior  with  blue  litmus  and 
an  alkaline  with  red.  In  mother's  milk  the  alkaline  reaction  is  stronger 
than  in  cow's  milk,  but  the  attempts  to  determine  it  by  titration  with 
the  usual  indicators  lead  to  results  of  relatively  little  value  because  of 
the  disturbing  action  of  the  proteins  present.  The  salts  of  mother's 
milk  are  lower  than  in  cow's  milk. 

Analyses.  The  analysis  of  human  milk  seems  to  present  several 
points  of  difficulty  and  the  published  results  do  not  show  very  good 
agreement.  The  separation  of  the  proteins  offers  the  greatest  diffi- 
culty, as  the  simple  and  accurate  methods  employed  in  the  analysis  of 
cow's  milk  fail  to  give  equally  satisfactory  results  when  applied  to 
mother's  milk.  The  explanation  of  this  will  be  given  below.  There 
are  variations  in  the  composition  of  human  milk  as  in  that  from  other 
species,  but  average  values  are  about  as  given  below : 

Water    87.5 

Fat   3-8 

Casein   1 .6 

Albumin    0.5 

Sugar    6.2 

Salts    0.4 

100.0 


246  PHYSIOLOGICAL    CHEMISTRY. 

This  analysis  must  be  accepted  as  representing  the  facts  only  in  a 
general  way.  Indeed,  some  authors  go  so  far  as  to  assert  that  no  mean 
value  for  woman's  milk  is  possible,  as  the  variations  from  individual 
to  individual  are  too  great  to  permit  an  average  result  to  have  any 
legitimate  meaning.  This  much,  however,  is  well  established :  the  fat 
in  woman's  milk  is  not  greatly  different  in  amount  from  that  in  cow's 
milk;  the  sugar  is  about  fifty  per  cent  higher  in  the  mean;  the  salts 
are  lower,  sometimes  as  little  as  0.2  or  0.3  per  cent  of  ash  being  found; 
the  total  proteins  are  about  half  as  much  as  in  cow's  milk.  But  as  to 
the  relation  of  the  casein  to  the  albumin  and  as  to  the  nature  of  the 
casein  itself,  the  greatest  divergence  of  views  exists.  Some  analysts 
have  actually  found  more  albumin  than  casein  as  a  result  of  experi- 
ments. This  is  probably  due  to  the  employment  of  a  faulty  method 
for  the  precipitation  of  the  casein;  it  has  been  pretty  well  established 
that  the  conditions  of  precipitation  or  coagulation  are  entirely  different 
from  those  obtaining  for  cow's  milk.  It  is  indeed  likely  that  the  pro- 
tein called  casein  in  woman's  milk  is  quite  distinct  from  that  of  cow's 
milk.  Under  the  action  of  rennet  the  former  coagulates  in  fine  flakes 
while  the  curd  of  cow's  milk  as  at  first  produced  is  in  very  large  flakes. 
The  two  caseins  have  apparently  different  contents  of  sulphur  and 
phosphorus  and  give  up  their  nitrogen  in  digestion  experiments  in 
different  ways.  It  has  been  recently  suggested  that  the  product  coagu- 
lated as  casein  from  human  milk  may  contain  other  proteins  in  sufficient 
amount  to  give  it  the  peculiar  properties  noticed. 

It  must  be  remembered  that  the  salts  put  down  in  the  analysis  of 
milk  are  always  obtained  as  ash  from  the  incineration  of  an  evaporated 
residue.  In  the  original  milk  they  do  not  occur  in  this  form,  but  in 
part,  at  least,  in  organic  combination.  Most  of  the  sulphur  and  phos- 
phorus occur  in  this  condition.  The  lower  casein  content  of  mother's 
milk  must  be  responsible  for  part  of  the  salt  difference. 

Modified  Milk.  From  all  this  it  is  evident  that  attempts  to  modify 
cow's  milk  so  as  to  make  it  resemble  mother's  milk  must  be  more  or 
less  abortive,  as  we  are  not  able  to  duplicate  the  unknown  proteins  in 
the  human  secretion.  However,  many  suggestions  have  been  made  in 
this  direction  and  the  line  followed  is  essentially  this:  Cow's  milk  is 
first  diluted  with  an  equal  volume  of  water  or  whey  to  reduce  the 
proteins  to  the  proper  percentage  amount.  Then  a  certain  volume  of 
cream  is  added  to  restore  the  fat,  and  enough  milk  sugar  or  cane  sugar 
to  bring  that  constituent  up  to  about  6  per  cent.  Unfortunately,  the 
addition  of  fat  is  uncertain  because  of  the  great  variations  in  market 
cream.     Good  cream  should  contain  at  least  20  per  cent  of  fat,  but  is 


MILK.  247 

usually  much  inferior  to  this  standard.  The  cream  sold  in  cities  often 
contains  from  5  to  10  per  cent  of  fat  only.  Assuming,  however,  a 
cream  containing  20  per  cent  of  fat,  3.5  per  cent  of  casein  and  3.5  per 
cent  of  sugar,  and  taking  the  gram  and  cubic  centimeter  as  equivalent 
for  our  purpose,  the  following  illustration  will  serve  as  an  example 
of  such  a  modification.  Starting  with  average  market  milk  of  the 
composition  given  some  pages  back,  500  cc.  may  be  mixed  with  water, 
cream  and  sugar  to  give  a  result  as  follows : 


In  500  cc.  of  Market 
Milk. 

1000  cc.  contains,  approximately,  after  addition  of  400  cc. 
of  Water,  100  cc.  of  Cream,  35  gm.  of  Milk  Sugar. 

Fat 

17-5  gm. 
22.5 

19-5 
3-5 

37-5  gm- 
61.0 

23.0 
4.0 

3.8  per  cent. 
6.1 

Sugar 

Proteins 

2-3 

0.4 

Salts 

This  mixture  has  a  percentage  composition  pretty  close  to  that  of 
mother's  milk.  Sometimes  the  dilution  is  made  with  whey  in  place  of 
water ;  the  final  result  in  this  case  is  a  product  containing  a  little  more 
protein  because  of  the  content  of  albumin  in  the  whey. 

Another  important  distinction,  however,  must  not  be  lost  sight  of. 
While  the  milk  of  the  cow  is  sterile  when  it  leaves  the  udder  it  takes 
up  from  the  hands  of  the  milker  or  from  the  air  a  large  number  of 
bacteria  which  speedily  increase  to  give  a  content  of  millions  to  the 
cubic  centimeter.  Most  of  these  are  doubtless  harmless  and  have  no 
bad  effect  on  the  milk ;  of  others  this  cannot  be  said,  as  their  presence 
soon  leads  to  changes  in  the  milk  which  may  render  it  absolutely  unfit 
for  use  as  an  infant  food.  Mother's  milk  is  and  remains  sterile  and 
is  therefore  free  from  this  danger.  It  must  be  recalled  further  that 
the  bactericidal  behavior  of  human  milk  is  relatively  very  strong. 
While  all  milks  seem  to  have  a  certain  content  of  bacteriolysins,  these 
anti  bodies  in  mother's  milk  are  most  potent  as  far  as  the  destruction 
of  the  ordinary  bacteria  is  concerned.  It  is  quite  likely  that  no  small 
portion  of  the  superiority  of  human  milk  as  infant  food  is  due  to  this 
observed  fact. 

All  kinds  of  milk  are  affected  to  some  extent  by  peculiar  flavoring 
or  other  accidental  substances  in  the  food  of  the  parent  animal.  It  is 
a  well-known  fact  that  cows  having  access  to  certain  weeds  yield  a 
milk  with  characteristic  taste  and  odor.  In  the  same  way  many  sub- 
stances given  as  remedies  pass  to  some  extent  into  the  milk  of  the 
mother  and  may  have  an  effect  on  the  nursing  child.  Bay  rum  used 
for  bathing  the  breasts  of  a  nursing  mother  has  been  known  to  pass, 
in  part  at  least,  into  the  milk  and  give  to  it  a  very  strong  odor  and  taste. 


248  PHYSIOLOGICAL   CHEMISTRY. 

THE  MILK  OF  OTHER  ANIMALS. 

In  some  countries  the  milk  of  the  goat  and  the  ass  have  economic 
importance,  and  mare's  milk  is  used  by  certain  Asiatic  peoples  in  pro- 
ducing a  fermented  beverage.  Analyses  of  several  kinds  of  milk  are 
on  record;  some  of  these  are  given  in  the  following  table,  taken  mainly 
from  the  Konig  compilation : 


Goat. 

Ass. 

Mare. 

Sow. 

Bitch. 

Cat. 

Sheep. 

Elephant. 

Water 

86.9 
4.1 

4.4 

3-7 
0.9 

90.O 
1-3 
6.3 
2.1 

0.3 

90.0 
I.I 
6.7 
1.9 
0.3 

82.4 
6.4 
4.0 
6.1 
1.1 

75-4 

9.6 

3-i 

11. 2 

0.7 

8l.6 
3-4 
4-9 
9-4 
0.7 

81.3 

6.8 

4-7 
6.4 
0.8 

67.O 

Fat 

22.0 

Sugar  

7-4 

Proteins 

3.0 

Salts 

0.6 

Bunge  has  called  attention  to  a  relation  which  exists  between  the 
composition  of  a  milk  and  the  rapidity  of  growth  of  the  animal  feeding 
on  it.  In  the  case  of  the  young  of  the  dog,  cat  and  sheep,  for  example, 
the  rate  of  growth  immediately  after  birth  is  very  rapid  and  the  milk 
of  the  mothers  correspondingly  rich  in  proteins  and  calcium  phosphate. 
The  young  of  the  horse  and  ass  are  slow  growers,  that  is,  a  relatively 
long  period  is  required  for  them  to  double  in  weight.  These  mothers' 
milks  are  low  in  proteins  and  salts,  but  relatively  high  in  sugar.  In 
the  human  species  these  relations  are  even  more  pronounced. 


CHAPTER    XVII. 
THE  CHEMISTRY  OF  THE  LIVER.    BILE.    CELLS  IN  GENERAL. 

From  the  earliest  days  of  physiological  chemical  investigation  the 
composition  of  the  liver  cells  and  the  nature  of  the  processes  taking 
place  there  have  been  the  subject  of  many  studies.  It  is  well  known 
that  the  liver  has  a  certain  definite  work  to  do  in  the  animal  organism 
and  of  some  of  the  functions  we  have  fairly  accurate  ideas.  Of  other 
functions  there  is  much  yet  in  dispute,  but  it  may  be  said  that  a  number 
of  synthetic  reactions  are  unquestionably  carried  out  through  the 
activity  of  cell  enzymes  there  formed.  Before  taking  up  the  special 
work  of  the  liver  cells  something  should  be  said  of  the  composition  of 
animal  cells  in  general. 

COMPOSITION  OF  CELLS. 

In  structure  all  animal  cells  agree  in  consisting  of  two  essential  parts, 
a  nucleus  and  surrounding  protoplasm.  In  young  cells  these  two  parts 
are  usually  easily  recognized,  but  in  the  old  cells  of  complex  structures 
they  assume  various  forms,  bearing  apparently  little  resemblance  to  the 
original  type.  Cells  are  in  general  the  center  of  the  various  chemical 
reactions  taking  place  in  the  body.  Some  of  these  reactions  take  place 
in  the  fluids  outside  the  cell,  but  by  the  aid  of  ferments  of  cell  origin; 
most  reactions,  however,  seem  to  be  carried  on  within  the  cell,  which 
may  be  illustrated  by  the  familiar  conversion  of  sugar  into  alcohol  and 
carbon  dioxide  by  the  yeast  cell.  The  enzyme  which  does  this  work 
may,  however,  be  extracted,  as  has  been  already  shown. 

Of  the  chemical  nature  of  nucleus  and  protoplasm  not  a  great  deal 
is  known.  It  is  extremely  difficult  to  isolate  original  cells  from  their 
modified  products  or  tissues  in  general,  and  a  sharp  chemical  differ- 
entiation between  the  two  component  parts  of  the  cells  is  not  yet  pos- 
sible, however  simple  the  microscopic  differentiation  may  be.  But 
some  points  have  been  worked  out  and  these  will  be  briefly  referred  to. 
Our  information  here  comes  mainly  from  analyses  of  the  simplest  cells, 
as  in  cells  of  the  more  complex  tissues  the  true  cell  characteristics  are 
obscured. 

The  Nucleus.  The  most  important  chemical  constituent  of  the 
nucleus  is  the  complex  protein  substance  known  as  nuclcin,  already 

249 


25O  PHYSIOLOGICAL    CHEMISTRY. 

referred  to  in  an  earlier  chapter.  Nucleins  of  different  character  are 
obtained  from  different  sources.  These  nucleins  exist  in  combination 
as  nucleo-proteids,  and  in  turn  break  up  into  nucleic  acids  and  a  protein 
fraction,  which  was  explained  in  the  chapter  referred  to.  The  cell 
nucleus  appears  to  consist  very  largely  if  not  wholly  of  the  nucleo- 
proteid.  On  digestion  with  pepsin  and  hydrochloric  acid  the  nuclein 
is  separated  and  may  be  purified  by  washing  with  water,  dissolving  in 
very  weak  alkali  and  reprecipitating  with  acid.  By  digestion  with 
pancreatic  extract  the  nucleic  acid  is  left.  The  pure  nuclein  is  a  white 
amorphous  substance  which  gives  the  Millon  test  and  the  biuret  test. 
The  various  nuclein  substances  in  cells  are  characterized  by  a  strong 
affinity  for  dye  stuffs,  especially  for  some  of  the  coal  tar  dyes;  this 
property  is  utilized  in  the  microscopic  examination  of  tissues.  Nuclein 
fused  with  sodium  carbonate  and  nitrate  yields  phosphate,  but  heated 
without  the  alkali  an  acid  residue  (metaphosphoric  acid)  is  left.  Of 
other  constituents  of  the  nucleus  but  little  is  known.  Lecithin  may  be 
present. 

By  various  decompositions  nuclein  substances  yield  a  number  of 
peculiar  basic  bodies  known  as  the  xanthine  or  purine  bases,  which  will 
be  considered  in  a  following  chapter.  The  cell  nucleus  contains  in 
combination  a  number  of  metallic  elements  among  which  iron  is  perhaps 
the  most  important.  Potassium  salts  are  present,  while  those  of 
sodium  are  present  only  in  traces,  if  at  all. 

The  Protoplasm.  This  soft  spongy  portion  of  the  cell  consists 
largely  of  water.  The  solid  part,  making  up  10  to  20  per  cent  usually, 
contains  several  albumins  proper  and  nucleo-proteids.  Lecithin  is  an 
important  and  relatively  abundant  constituent  of  the  protoplasm.  Its 
presence  seems  to  be  intimately  associated  with  phenomena  of  repro- 
duction and  building  up  of  new  tissues.  The  chemistry  of  lecithin,  as 
a  phosphoric  acid  fat,  has  been  explained  already,  and  it  must  be  re- 
called that  this  term  includes  a  number  of  phosphatides.  In  the  cell 
protoplasm  they  doubtless  exist  in  part  as  a  complex  lecitho-protein. 
This  may  account  for  the  fact  that  the  separation  of  the  lecithin  is 
sometimes  difficult. 

In  all  cases  the  protoplasm  seems  to  contain  the  complex  alcohol 
substance  cholesterol,  glycogen  and  ordinary  fats.  How  these  various 
complexes  exist,  to  what  extent  they  are  necessary  or  essential  in  the 
cell  structure,  we  can  not  say.  As  cells  have  various  functions  to  per- 
form, they  have  the  power  of  producing  different  ferments  for  the 
purpose  and  such  products  can  not  be  distinguished  by  our  present 
methods  of  analysis  from  the  material  of  the  cell  itself. 


THE    CHEMISTRY    OF    THE    LIVER.  25 1 

FUNCTIONS  OF  THE  LIVER  CELLS. 

The  anatomical  location  of  the  liver  gives  it  a  most  important  rela- 
tion to  the  other  organs  of  the  body.  With  the  exception  of  the  fats 
most  of  the  products  of  the  digestion  of  foods  pass  through  the  portal 
vein  into  the  liver  and  there  undergo  certain  preparatory  changes. 
Substances  not  true  foods  take  the  same  course  and  many  toxic  bodies, 
metallic  and  alkaloidal,  find  a  resting  place  in  the  liver.  In  toxico- 
logical  examinations  the  liver,  after  the  stomach,  is  the  most  important 
organ  for  analysis. 

Xot  only  are  the  fundamental  food  stuffs,  the  proteins  and  the  carbo- 
hydrates, worked  over  and  more  or  less  altered  in  the  liver,  but  partly 
metabolized  products  seem  to  be  further  changed  in  passing  through 
this  organ  and  are  there  brought  into  a  condition  for  final  excretion. 
The  evidences  that  such  reactions  take  place  have  been  worked  out  in  a 
number  of  cases  experimentally  and  will  be  referred  to  below. 

Composition  of  the  Liver.  We  have  here  the  materials  found  in 
cells  in  general  and  also  others  having  special  functions.  The  protein 
substances  separated  belong  to  several  groups ;  albumin,  globulin  and  a 
nucleo-proteid  have  been  recognized.  Iron  exists  in  combination  with 
several  of  these  protein  bodies.  One  of  these  is  known  as  ferratin  and 
contains  the  iron  in  complex  combination;  others  appear  to  be  albu- 
minates in  which  the  iron  is  more  readily  recognized. 

Next  to  the  proteins  the  fats  are  relatively  abundant  in  the  liver  and 
may  amount  to  3  or  4  per  cent  by  weight  normally.  Pathologically,  by 
fatty  degeneration,  or  by  filtration  from  other  tissues,  the  fat  may  be 
greatly  increased,  even  to  30  or  35  per  cent  of  the  weight  of  the  whole 
organ.  The  liver  fat  is  usually  comparatively  soft,  but  that  formed  in 
some  degenerations  is  harder. 

The  proportion  of  lecithins  in  the  liver  is  variable  and  is  ordinarily 
below  the  true  fats.  The  average  amount  is  from  2  to  3  per  cent.  It 
has  a  more  important  function  to  perform  than  have  the  fats  proper, 
since  it  is  found  by  experiment  that  in  starvation  the  lecithin  fat  is  the 
last  to  disappear.  The  ether  extract  of  the  organ  in  this  case  is  largely 
lecithin. 

Much  has  been  written  of  the  functions  of  the  lecithin  bodies,  and 
part  of  their  behavior  is  possibly  physical.  Recent  investigations  have 
suggested  that  in  certain  ferment  phenomena  they  may  play  the  part  of 
activators  for  the  pro-ferments.  In  the  liver,  where  a  multiplicity  of 
such  reactions  occur,  the  presence  of  such  large  quantities  of  these 
phosphatides  may  have  a  special  meaning. 

The  most  important  substance  found  in  the  liver  is  probably  glyco- 


252  PHYSIOLOGICAL    CHEMISTRY. 

gen,  which  is  a  transformation  product  and  variable  in  quantity.  The 
amount  present  at  any  one  moment  depends  on  the  carbohydrate  con- 
sumption and  the  time  which  has  elapsed  since  a  meal.  It  may  be 
as  high  as  15  per  cent  of  the  whole  weight  of  the  organ  or  may  run 
down  to  a  fraction  of  1  per  cent,  after  fasting  or  after  the  performance 
of  hard  work.     The  formation  of  glycogen  will  be  discussed  below. 

The  liver,  consisting  largely  of  cells  in  rapid  state  of  change,  fur- 
nishes a  relatively  large  amount  of  the  so-called  nitrogenous  extractives. 
These  include  the  xanthine  and  related  bodies,  urea,  uric  acid,  leucine, 
cystin  and  other  substances  representing  certain  stages  in  metabolism. 
The  total  amount  of  these  compounds  present  at  any  one  time  is  very 
small,  and  probably  not  over  0.5  per  cent  of  the  dried  organ;  but  even 
this  small  amount  is  important,  as  will  appear  below. 

We  have  finally  several  mineral  substances  present.  These  include 
essentially  the  chlorides  and  phosphates  of  the  alkali  and  alkali  earth 
metals  with  some  iron  compounds.  Of  the  latter  those  with  proteins 
have  been  mentioned  above,  but  other  iron  salts  are  present  and  the 
quantity  may  be  increased  by  the  administration  of  inorganic  substances 
as  remedies.  In  normal  conditions  the  iron  content  is  extremely  var- 
iable. The  amount  may  be  accurately  determined  only  after  washing 
out  the  blood  (containing  hemoglobin)  by  aid  of  salt  solution  of  proper 
strength,  about  0.9  per  cent.  Recent  investigations  have  shown  that 
in  the  livers  of  women  the  iron  varies  from  0.05  per  cent  to  0.09  per 
cent  of  the  dry  substance,  while  in  men  the  content  is  more  irregular, 
running  from  0.05  per  cent  to  0.37  per  cent.  The  amount  seems  to 
increase  with  age,  but  no  explanation  for  the  variations  can  be  given. 
In  children  and  very  young  animals  the  content  is  also  high.  It  sinks, 
and  rises  again,  later  in  life.  In  addition  to  the  iron  a  trace  of  copper 
is  said  to  be  always  present  and  may  have  some  physiological  function. 
Other  metals  occasionally  found  are  probably  of  accidental  occurrence, 
as  the  liver  retains  such  foreign  substances  through  a  long  period. 

CHEMICAL  CHANGES   IN  THE  LIVER. 

In  recent  years  much  has  been  written  on  this  obscure  but  highly 
important  topic.  Many  of  the  changes  taking  place  in  the  liver  come 
under  the  head  of  fermentations,  enzymic  reactions.  Hofmeister 
pointed  out,  a  number  of  years  ago,  that  there  are  at  least  eleven  of 
these  in  play.  He  mentioned  a  proteolytic  and  a  nuclein-splitting  fer- 
ment, one  which  splits  off  ammonia  from  amino  acids,  a  rennet  ferment, 
a  fibrin  ferment,  an  autolyzing  ferment,  a  bactericidal  ferment,  an 
oxydase,  a  lipase,  a  maltase  and  a  glucase.     But  since  then  our  views 


THE    CHEMISTRY    OF    THE    LIVER.  253 

have  been  much  broadened.  We  have,  in  addition  to  these  reactions, 
which  result  in  general  in  the  breaking  down  of  molecules,  a  number 
of  others  which  are  synthetic  in  their  nature.  A  brief  study  of  what 
is  known  of  all  these  changes  is  sufficient  to  indicate  the  immense 
importance  of  the  liver  in  the  metabolic  phenomena  of  the  body. 

CARBOHYDRATE  CHANGES. 

These  reactions  will  be  considered  first  because  they  have  been  the 
most  thoroughly  studied  and  also  because  of  their  intrinsic  importance. 

Glycogen  Formation.  It  was  long  ago  established  that  the  food 
carbohydrates  after  digestion  reach  the  circulation  almost  exclusively 
by  way  of  the  portal  vein  and  the  liver.  In  the  normal  food  of  man 
and  the  herbivora  the  carbohydrate  food  is  usually  starch  and  this 
becomes  dextrin,  maltose  and  finally  glucose  before  absorption.  As  no 
marked  accumulation  of  the  sugar  takes  place  in  the  blood  after  a  meal 
it  must  follow  that  it  or  some  derived  reserve  product  must  be  tem- 
porarily retained  somewhere.  The  place  of  this  retention  is  the  liver 
and  the  form  in  which  the  sugar  is  held  is  glycogen.  The  chemical 
reactions  of  glycogen  have  been  discussed  in  the  chapter  on  the  carbo- 
hydrates, but  in  this  place  other  relations  must  be  considered.  No 
simple  answer  can  be  given  to  the  question  as  to  the  method  of  forma- 
tion of  glycogen  from  sugar.  Although  the  formula  is  commonly 
written  C6H10O5,  it  is,  like  common  starch,  certainly  a  multiple  of  this. 
Hence  a  simple  equation  connecting  glucose  and  glycogen  of  the  form 

CeH1206  —  H2O  =  C6H,0O5 

is  not  strictly  correct.  Besides,  several  other  facts  appear  which  com- 
plicate the  problem.  While  glucose  is  ordinarily  the  sugar  which 
passes  through  the  portal  vein,  other  sugars  are  also  consumed  and  in 
the  digestive  process  do  not  become  changed  to  glucose.  From  cane 
sugar  we  have  some  fructose  and  from  milk  sugar  some  galactose,  and 
with  these  in  the  food  it  appears  that  glycogen  is  still  formed.  More- 
over, it  has  been  shown  that  substances  not  carbohydrate  at  all  may 
give  rise  to  glycogen.  Animals  have  been  starved  until  the  liver  was 
practically  free  from  glycogen  (as  known  by  previous  trials  with  other 
animals)  and  then  fed  on  fibrin  or  washed  out  lean  meat.  On  killing 
the  animals  a  short  time  later  a  store  of  glycogen  was  found  in  the 
liver,  indicating  its  formation  from  something  in  the  protein.  With 
such  facts  in  mind  it  is  not  possible  to  form  any  simple  theory  of  the 
production  of  the  reserve  substance.  From  the  sugars  it  is  likely  that 
some  such  reaction  takes  place  as  occurs  in  the  formation  of  starch  in 


254  PHYSIOLOGICAL    CHEMISTRY. 

plants.  The  carbohydrate  built  up  in  the  plant  from  water  and  car- 
bonic acid  is  a  sugar  and  this  is  transformed  by  some  enzymic  reaction 
into  starch  as  a  reserve  material.  The  mechanism  of  this  change,  how- 
ever, is  quite  obscure. 

Attempts  have  been  made  to  connect  the  formation  from  proteins 
with  the  sugar  group  of  the  gluco-proteids,  but  casein  and  gelatin  fed 
to  animals  lead  also  to  production  of  glycogen,  and  these  bodies  in  pure 
condition  do  not  furnish  a  sugar  complex  by  laboratory  treatment.  In 
addition  to  this  it  is  impossible  that  the  sugar  group  could  be  abundant 
enough  in  the  other  common  proteins  to  account  for  the  large  amount 
of  glycogen  which  may  be  formed  by  protein  diet.  These  facts  lead 
to  the  view  that  a  synthesis  must  be  concerned  in  the  reaction.  Such 
protein  derivatives  as  leucine,  the  hexone  bases  and  other  bodies  have 
been  thought  of  as  leading  possibly  to  the  end,  but  direct  experiments 
with  animals  have  given  no  satisfactory  proof  of  such  a  hypothesis. 
For  the  present,  therefore,  the  method  of  production  from  proteins 
must  be  left  without  explanation. 

A  diet  of  fat  leads  also  to  glycogen  accumulation  or  formation  in 
small  amount,  according  to  some  recent  observations.  This  latter 
reaction  requires  some  kind  of  an  oxidation  and  is  more  difficult  of 
explanation  than  the  other.  It  must  be  remembered  that  an  accumu- 
lation of  glycogen  may  follow  from  diminished  destruction  as  well  as 
from  increased  production,  and  where  the  amount  in  question  is  small, 
an  apparent  increase  may  be  traced  to  errors  of  observation  or  experi- 
ment. In  a  mixed  diet  it  is  practically  impossible  to  trace  the  effect 
of  any  one  substance.  The  behavior  of  pentoses  is  an  illustration; 
according  to  the  statements  of  some  authors  these  carbohydrates  in- 
crease glycogen.  It  may  be,  however,  that  they  simply  behave  as 
sparers  of  glycogen  by  undergoing  oxidation,  which  otherwise  the 
glycogen  would  have  to  undergo. 

Not  all  the  carbohydrate  reaching  the  portal  vein  is  transformed  in 
the  liver;  apparently  only  a  certain  portion  is  so  changed,  while  the 
excess  is  stored  up  temporarily  in  other  organs.  This  is  evident  from 
the  fact  frequently  observed  in  animal  experiments  that  the  amount  of 
glycogen  in  the  liver  is  below  what  should  be  expected  from  the  food 
when  this  is  excessive  in  carbohydrates.  With  ordinary  or  deficient 
feeding  the  liver  doubtless  is  able  to  store  as  glycogen  all  the  sugar  con- 
veyed to  it,  but  an  excess  must  find  lodgment  elsewhere.  The  muscles 
undoubtedly  receive  the  greater  share  of  this  excess.  In  extreme  cases 
the  liver  may  hold  200  grams  of  glycogen,  which  would  correspond  to 
the  same  weight  of  starch. 


THE    CHEMISTRY    OF    THE    LIVER.  255 

Glycogen  Destruction.  This  stored  up  glycogen  disappears  in 
normal  conditions  gradually  after  its  accumulation;  the  disappearance 
is  hastened  by  work  or  by  lowering  of  temperature,  showing  that  it 
may  be  called  upon  for  supply  of  heat  as  well  as  for  direct  mechanical 
work.  Before  being  utilized,  however,  for  these  purposes  the  glycogen 
must  be  thrown  back  into  the  form  of  sugar;  how  this  is  done  is  still 
a  matter  of  discussion.  According  to  one  view  the  action  is  a  "  vital  " 
one,  depending  on  the  life  of  the  cells  of  the  liver  themselves;  by 
another  view  this  conversion  is  wholly  enzymic,  a  peculiar  ferment 
bringing  about  the  change  during  life  as  well  as  post-mortem.  This 
question  has  lost  much  of  its  importance  since  the  work  of  Buchner  on 
the  zymase,  or  enzyme  of  yeast  active  in  alcohol  formation,  as  we  now 
know  that  enzymes  are  present  where,  by  earlier  methods  of  experi- 
ment, they  were  supposed  to  be  absent. 

The  more  recent  careful  experiments  seem  to  show  beyond  question  that  a  true 
glycogen-splitting  ferment  is  present.  By  proper  manipulation  the  cell  effect  may  be 
excluded,  while  that  of  the  ferment  is  left  intact.  This  may  be  accomplished  in 
the  following  way :  The  fresh  organ  is  washed  free  from  blood  by  forcing  water 
through  the  portal  vein  until  that  escaping  by  the  hepatic  veins  is  clear  and  colorless. 
The  liver  is  then  chopped  fine  and  allowed  to  stand  a  day  in  a  large  excess  of 
alcohol  for  dehydration.  The  alcohol  is  poured  off,  the  residue  pressed,  dried  at 
a  low  temperature  and  ground  to  a  powder.  In  this  form  it  is  suitable  for  extrac- 
tion with  something  which  does  not  interfere  with  enzymic  power,  but  which  pre- 
vents bacterial  or  other  cell  activity.  For  this  purpose  chloroform  water,  or  solu- 
tions of  sodium  fluoride  have  been  used.  A  good  extracting  mixture  may  contain 
in  100  cc.  of  water  0.2  gm.  of  sodium  fluoride  and  0.9  gm.  of  sodium  chloride. 
The  liver  powder  is  exhausted  with  such  a  solution  at  a  temperature  of  380  and  the 
filtered  liquid  obtained  may  be  used-  in  two  ways.  On  standing,  the  sugar  in  the 
solution  increases  while  the  glycogen  decreases.  In  addition,  if  pure  glycogen  be 
added  to  such  an  extract  it  is  found  also  to  diminish  with  corresponding  increase 
of  sugar.  It  is  further  found  that  boiling  the  fluoride  extract  destroys  all  con- 
verting power,  which  fact  speaks  likewise  for  enzyme  action. 

The  glycogen-converting  power  of  solutions  made  as  above  is  considerable  and 
sufficient  to  fully  account  for  the  post-mortem  increase  of  sugar  always  found  in 
the  liver.  By  extracting  not  the  whole  liver  but  portions  it  is  possible  to  compare 
the  distribution  of  the  ferment.  Experiments  made  with  this  end  in  view  have 
shown  that  this  is  pretty  uniform.  By  following  the  same  general  method  Pick 
has  compared  the  ferment  activity  of  the  liver  with  that  of  other  organs  where 
glycogen  may  be  stored.  In  such  experiments  the  diastatic  action  of  the  liver  has 
been  found  to  be  in  excess  as  should  be  expected,  since  this  is  the  organ  where 
the  greatest  accumulation  normally  takes  place.  This  normal  conversion  of  gly- 
cogen by  the  liver  ferment  is  interfered  with  by  various  substances  which  may  be 
taken  as  remedies;  quinine  salts  seem  to  be  especially  active. 

AUTOLYTIC  FERMENTATION. 
The  liver,  or  other  organ,  removed  from  the  body  and  left  to  itself 
speedily  undergoes  a  change.     Unless  precautions  are  taken  to  prevent 


256  PHYSIOLOGICAL    CHEMISTRY. 

it  the  bacterial  decomposition  may  become  pronounced  and  obscure 
other  reactions.  Some  years  ago  Salkowski  gave  the  name  auto- 
digestion  to  the  fermentations  taking  place  in  the  liver,  in  which  a 
change  in  the  nitrogenous  constituents  is  mainly  involved.  Other 
chemists  followed  the  subject  further,  taking  precautions  to  exclude 
all  bacterial  influences,  and  have  brought  to  light  a  number  of  very 
peculiar  reactions  which  follow  from  the  presence  of  ferments  in  the 
organs.  The  name  autolysis  has  been  given  to  these  self -digestion 
reactions  in  general.  They  are  not  confined  to  the  liver,  but  are 
observed  in  all  organs.  An  enormous  literature  has  accumulated 
already  on  this  topic,  because  it  has  great  practical  as  well  as  scientific 
importance.  In  these  spontaneous  digestions  various  products  are 
formed,  some  of  which  are  volatile;  a  general  softening  of  the  tissues 
concerned  may  also  take  place  and  the  sum  of  these  changes  is  impor- 
tant in  bringing  about  the  difference  between  fresh  meat,  and  stored, 
"  ripe  "  or  "  hung  "  meat,  for  example.  While  bacteria  play  an  impor- 
tant part  in  curing  meat  it  is  well  known  that  changes  go  on  within  the 
tissues  which  cannot  be  due  to  bacterial  action.  These  are  the  autolytic 
changes  which  were  first  clearly  followed  in  the  liver,  and  which  will 
be  here  briefly  discussed. 

The  Production  of  Organic  Acids.  This  is  one  of  the  simplest 
phenomena  observed.  If  the  livers  of  dogs  or  other  animals  are  care- 
fully removed  and  kept  under  chloroform  or  toluene  a  gradual  gain  in 
acidity  is  observed.  The  liver  must  be  minced  before  being  covered 
with  the  protecting  liquid.  The  autolysis  in  this  case  is  slow,  weeks 
or  months  being  required  to  show  any  large  amount  of  acid.  The  best 
temperature  for  the  experiment  is  38-400  C.  Instead  of  employing 
antiseptics  it  is  possible  with  care  to  remove  and  store  the  liver  in  sterile 
jars  aseptically.  Under  these  conditions  the  spontaneous  change  is 
very  rapid,  more  acid  being  formed  in  one  day,  ordinarily,  than  after 
a  month  of  the  antiseptic  treatment.  By  making  several  pieces  of  the 
liver  on  removal  from  the  animal,  putting  each  in  a  separate  jar  and 
testing  one  from  time  to  time,  it  is  possible  to  follow  the  course  of  the 
autolysis.  Among  the  acids  produced  formic,  acetic,  fermentation 
lactic  and  paralactic,  butyric  and  succinic  have  been  recognized.  In 
experiments  described  by  Magnus-Levy  the  total  acid  formed  in  one 
day  in  100  grams  of  liver,  by  the  aseptic  treatment,  may  correspond  to 
over  20  cc.  of  normal  alkali.  If  this  were  calculated  as  lactic  acid  it 
would  amount  to  1.8  gm.  The  relation  between  the  volatile  and  non- 
volatile acids  varies  with  the  animal,  but  not  regularly. 

It  is  not  possible  to  trace  exactly  the  source  of  all  these  acids,  but 


THE    CHEMISTRY    OF    THE    LIVER.  257 

apparently  they  come  in  part  from  a  decomposition  of  the  sugar  of  the 
liver,  since  this  is  found  to  decrease  as  the  acid  increases.  Lactic  acid 
may  be  formed  first  from  sugar  and  butyric  acid  from  the  lactic  as  in 
the  bacterial  fermentations.  The  appearance  of  hydrogen  and  carbon 
dioxide  at  the  same  time  favors  this  view. 

The  Alteration  in  the  Proteins.  When  subjected  to  aseptic  auto- 
digestion,  or  to  the  same  digestion  with  chloroform  or  toluene,  the 
protein  substances  gradually  break  down  into  simpler  products.  Among 
these  the  amino  acids  may  be  most  readily  recognized ;  there  is  also  an 
increase  in  the  nitrogen  which  may  be  distilled  off  with  magnesia.  The 
behavior  here  is  somewhat  similar  to  that  which  follows  in  acid  hydrol- 
ysis of  the  proteins,  or  which  occurs  in  prolonged  boiling  with  water 
under  pressure;  in  both  cases  a  kind  of  hydrolysis  results  and  this  may 
be  what  takes  place  in  auto-digestion. 

In  prolonged  aseptic  auto-digestion  of  the  liver  very  considerable 
quantities  of  leucine  and  tyrosine  are  formed;  on  the  outer  surfaces, 
where  evaporation  can  take  place,  the  latter  may  even  separate  in  crystal- 
line bunches  easily  recognized.  The  hexone  bases  and  bodies  of  the 
xanthine  group  also  result  but  not  always  in  very  great  quantities. 
The  greater  number  of  these  reactions  are  those  of  hydrolytic  cleavage, 
and  that  they  follow  spontaneously  is  one  of  the  best  proofs  of  the 
character  of  the  ferment  agents  present.  While  in  a  general  way  sim- 
ilar, it  has  been  found  that  certain  organs  yield  amino  acid  and  other 
groups  not  liberated  in  the  autolysis  of  other  organs.  This  is  an 
extremely  interesting  fact,  as  it  points  to  the  specificity  of  function 
suggested  also  by  other  reactions.  It  has  been  pointed  out,  further, 
that  the  corresponding  organs  in  different  animals  show  certain  differ- 
ences in  this  respect. 

Pathological  Importance.  This  possibility  of  self-digestion  in  the 
liver  and  other  organs  may  help  explain  some  of  the  phenomena 
observed  in  pathological  conditions.  The  acids  found  sometimes  in 
the  urine  as  well  as  the  leucine  and  tyrosine  have  usually  been  traced 
to  the  liver.  These  experiments  show  the  rapidity  with  which  such 
products  may  be  formed  by  a  degenerative  process.  Pathologically  the 
urine  sometimes  shows  a  very  high  reducing  power  which  cannot  be 
associated  with  sugar  or  uric  acid  or  creatinine.  The  liquid  formed  in 
the  liver  autolysis  is  always  strongly  reducing  in  action  and  this  may 
suggest  an  explanation  for  the  observation  of  the  urine. 

Bactericidal  Products.  It  is  worthy  of  note  that  in  these  autolytic 
decompositions  substances  are  formed  which  have  a  marked  bacteri- 
cidal action.  This  has  been  shown  in  many  ways  and  the  suggestion 
18 


258  PHYSIOLOGICAL    CHEMISTRY. 

appears  reasonable  that  in  the  continuous  breaking  down  processes 
going  on  in  various  organs  we  have  some  of  the  factors  of  natural 
immunity.  These  autolytic  products  must  not  be  confounded  with 
the  alexins  already  referred  to.  In  a  few  experiments  on  record  injec- 
tions with  pressed  out  juice  from  autolyzed  organs  have  been  sufficient 
to  prevent  death  in  small  animals  infected  with  virulent  cultures.  The 
bactericidal  action  of  the  fresh  liver  or  other  organ  is  comparatively 

slight. 

OTHER   FERMENT   ACTIONS. 

Other  ferments  present  in  the  liver  have  not  been  very  thoroughly 
studied.  The  presence  of  a  fat-splitting  ferment  or  lipase  has  been 
shown,  but,  as  yet,  little  is  definitely  known  of  the  extent  of  its  action 
in  the  body.  The  oxidase  ferments  are  better  known  and  the  action 
of  liver  extracts  in  bringing  about  oxidations  of  various  organic  sub- 
stances has  been  studied  with  the  object  of  throwing  some  light  on 
normal  oxidations  in  the  body.  How  many  of  these  oxidizing  fer- 
ments the  liver  may  contain  is  of  course  not  known.  The  reaction  thus 
far  the  most  carefully  studied  is  that  between  water  extracts  of  the 
liver  and  salicylic  aldehyde.     In  the  process  this  becomes  salicylic  acid. 

The  action  of  a  liquid  obtained  by  pressing  the  minced  liver  ground 
up  with  sand  has  been  studied  with  reference  to  its  power  of  hydro- 
lyzing  certain  esters.  Ethyl  butyrate  seems  to  be  readily  split  by  this 
liver  juice.  The  reaction  points  to  the  presence  of  a  lipase-like  fer- 
ment which  doubtless  has  the  power  of  splitting  other  bodies  of  this 
type.  The  boiled  liquid  is  without  the  ester-splitting  power.  It  has 
been  found  further  that  the  active  element  can  be  completely  salted 
out  by  addition  of  ammonium  sulphate  to  saturation,  and  it  may  be 
precipitated  by  addition  of  a  strong  solution  of  uranium  acetate. 

THE  BEHAVIOR   OF  THE  LIVER  WITH   POISONS. 

The  fact  has  been  referred  to  already  that  many  metallic  and  some 
organic  substances  combine  with  the  liver  cells.  All  this  has  a  prac- 
tical bearing  on  toxicological  investigations,  in  which  experience  has 
shown  the  importance  of  including  the  liver  in  the  analytical  tests. 
Recent  experiments  have  thrown  some  light  on  the  question  of  the 
manner  of  combination  of  poisons.  Corrosive  sublimate,  for  example, 
fed  in  very  small  portions  to  dogs  was  found  later  by  post-mortem 
examinations  in  the  globulin  fraction  of  the  liver  extract.  The  fixa- 
tion of  arsenic  is  different ;  it  combines  with  a  nuclein  substance  and  in 
very  stable  form,  which  explains  the  practical  difficulty  of  separating 
this  substance  in  forensic  investigations. 


THE    CHEMISTRY    OF    THE    LIVER.  259 

Experiments  have  also  been  published  showing  the  behavior  of  small 
doses  of  morphine  sulphate  and  strychnine  sulphate  in  the  liver.  It 
appears  that  the  retaining  power  of  the  liver  for  these  poisons  is  rela- 
tively large  when  they  are  administered  by  the  mouth  or  injected  into 
the  portal  vein.  The  retention  of  the  alkaloids  by  the  organ  has  been 
experimentally  shown.  Such  observations  have  an  important  bearing 
in  explaining  the  fact  that  many  poisons  are  far  more  active  when 
injected  hypodermically  than  when  given  through  the  stomach.  This 
seems  to  be  true  of  many  substances  besides  the  metallic  poisons  and 
the  alkaloids.  The  phenols,  for  example,  are  likewise  retained  to  a 
marked  extent  by  the  liver. 

SYNTHETIC  PROCESSES  IN  THE  LIVER. 

It  has  long  been  known  that  the  liver  is  the  seat  of  the  formation  of 
a  large  number  of  metabolic  products,  some  of  which  involve  syntheses. 
Several  of  these  reactions  may  be  briefly  explained  in  this  place,  but 
nothing  like  a  full  discussion  will  be  attempted.  A  few  illustrative 
cases  only  will  be  taken  to  show  in  a  general  way  what  is  best  known 
in  this  field.  The  reactions  mentioned  take  place  in  other  organs,  as 
well  as  in  the  liver,  but  as  the  latter  seems  to  be  mainly  concerned,  this 
is  a  good  place  to  discuss  them. 

The  Formation  of  Urea.  Of  all  the  synthetic  reactions  known  to 
occur  wholly  or  in  part  in  the  liver  this  one  has  been  the  most  thor- 
oughly studied.  The  older  notion  of  the  formation  of  urea  exclusively 
from  the  more  complex  uric  acid  is  no  longer  held ;  the  belief  that  the 
latter  complex  represents  a  portion  of  the  protein  residue  which  in 
some  manner  escaped  its  normal  and  proper  fate,  that  is,  conversion 
into  urea,  has  long  since  been  abandoned  in  view  of  much  accumulated 
evidence  to  the  contrary.  Indeed,  at  the  present  time  it  appears  more 
likely  that  a  part  of  the  uric  acid  excretion  may  be  traced  to  a  synthesis 
from  urea. 

A  great  many  observations  unite  in  suggesting  the  liver  as  the  organ 
in  which  urea  is  most  abundantly  produced,  and  certain  ammonium 
salts  as  being  largely  or  mainly  concerned  in  this  production.  These 
observations  have  been  made  in  the  laboratory  as  well  as  clinically.  In 
diseases  in  which  the  liver  is  involved  there  has  frequently  been  noticed 
a  marked  reduction  in  the  portion  of  the  excreted  nitrogen  appearing 
as  urea.  It  is  also  known  that  the  administration  of  ammonium  salts 
is  not  followed  by  an  increase  of  ammonia  in  the  urine.  Parallel  witli 
this  observation  we  have  the  further  one  made  on  the  blood,  which  has 
shown  that  the  fluid  of  the  portal  vein  is  far  richer  in  ammonia  than  is 


260  PHYSIOLOGICAL    CHEMISTRY. 

that  from  the  hepatic  vein.  Such  observations  have  been  followed  up 
by  experiments  in  which  fresh  blood  is  forced  through  a  living  or  a 
recently  removed  liver  by  means  of  specially  constructed  apparatus. 
The  same  blood  may  be  caused  to  pass  the  liver  many  times.  After 
passing  a  few  times  and  reaching  uniformity  in  composition  various 
ammonium  and  related  compounds  are  added  to  the  blood  and  the  cir- 
culation then  continued.  In  this  way  the  abundant  transformation  of 
ammonium  carbonate  into  urea  is  readily  shown.  It  has  also  been 
found  that  certain  amino  acids  are  converted  rather  readily  in  going 
through  the  liver.  Experiments  have  shown  that  in  the  course  of  a 
few  hours  several  grams  of  leucine,  glycocoll  or  aspartic  acid  may  be 
transformed  into  urea  under  these  unfavorable  conditions. 

The  importance  of  this  observation  will  be  recognized.  It  is  well 
known  that  the  amino  acids  are  among  the  most  important  of  the  dis- 
integration products  of  the  proteins ;  by  hydrolytic  and  other  cleavage 
reactions  these  amino  complexes  result,  and  we  see  here  the  possibility 
of  further  destruction  with  ultimate  formation  of  urea.  It  is  possible 
that  in  this  reaction  carbamates  are  concerned,  as  the  formation  of  urea 
by  alternate  oxidations  and  reductions  of  ammonium  carbamate  has 
been  shown  by  Drechsel.     These  reactions  illustrate  the  relations 

NH40  •  CO  •  NH2  +  O  =  NH20  •  CO  •  NH2  +  H20 

NH20-CO-NH2  +  H2=NH2-CO-NH2  +  H20 

It  has  been  shown  that  the  carbamic  acid  salt  frequently  appears  in 
urine,  and  perhaps  normally.     This  relation  is  also  apparent: 

NH4  — O  NH4  — O  NH2 

I  I  I 

CO      -*  CO+H20      -»      CO  +  H20 

NH4  — O  NH2—  I  NH2 

There  is  one  ferment  reaction  which  is  known  to  lead  to  the  forma- 
tion of  urea  under  definite  conditions,  and  this  is  the  production  from 
the  diaminic  acid  arginine.  The  liver  and  other  organs  contain  an 
enzyme,  known  as  arginase,  which  has  the  property  of  splitting  argi- 
nine into  urea  and  ornithine,  or  diamino  valeric  acid.  As  arginine  is 
known  to  be  produced  normally  by  the  erepsin  digestion  we  have  here 
a  source,  for  a  small  part  at  least,  of  the  urea  formation. 

The  Synthesis  of  Uric  Acid.  The  mode  and  place  of  the  forma- 
tion of  uric  acid  in  the  animal  organism  have  been  the  subjects  of 
numerous  investigations.  In  birds,  serpents  and  some  of  the  mammals 
the  excretion  of  nitrogen  is  largely  in  the  form  of  uric  acid,  and  experi- 
ments have  shown  that  it  is,  in  part  at  least,  of  synthetic  origin.     The 


THE    CHEMISTRY    OF    THE    LIVER.  26 1 

excretion  of  uric  acid  in  birds  is  increased  by  doses  of  ammonium 
salts ;  with  the  livers  extirpated  there  is  a  decrease  in  the  elimination 
of  uric  acid  and  increase  in  excretion  of  ammonium  compounds.  In  a 
number  of  such  observations  the  liver  has  been  connected  with  uric 
acid  formation,  and  transfusion  experiments,  in  which  blood  containing 
ammonium  lactate  and  certain  other  compounds  has  been  forced 
through  the  livers  of  geese,  pointed  to  the  same  kind  of  a  synthetic 
conversion.  For  the  higher  animals,  however,  a  different  formation 
has  usually  been  assumed,  the  oxidation  of  the  purine  bodies  coming 
from  the  breaking  down  of  nucleins  being  looked  upon  as  the  principal 
formative  reaction. 

Later,  in  a  chapter  on  the  urine,  the  relations  of  the  purines  to  uric 
acid  will  be  pointed  out.  It  is  sufficient  to  state  here  that  the  enzymic 
production  of  uric  acid  from  other  purines  has  been  clearly  shown  by 
recent  observers.  These  enzymes  are  contained  not  only  in  the  liver, 
but  in  the  spleen  and  elsewhere,  and  it  seems  likely  that  other  enzymes, 
which  have  been  called  nucleases,  must  begin  the  cleavage  of  the 
nucleins  or  parent  substances. 

Comparatively  recent  experiments  by  several  authors  suggest  syn- 
thetic reactions  as  likewise  possible.  Wiener,  for  example,  mixed 
chopped  beef  liver  with  physiologic  salt  solution  and  allowed  the  mix- 
ture to  stand  at  the  body  temperature  an  hour.  The  liquid  was  then 
pressed  out  and  the  uric  acid  in  it  determined  after  some  time  in  a 
given  volume.  To  the  same  volume  of  liver  extract  definite  weights 
of  urea  and  various  ammonium  and  sodium  salts  were  added  and  the 
mixture  allowed  to  stand  as  before.  In  certain  cases  a  very  marked 
increase  in  the  uric  acid  resulted,  pointing  to  the  presence  in  the  liver 
extract  of  some  agent  capable  of  effecting  the  combination.  The  best 
results  were  obtained  with  dialuric  acid  salts  and  tartronic  acid  and  urea. 

It  is  fair  to  state  that  another  interpretation  of  these  results  has  been 
given.  While  admitting  the  formation  of  uric  acid  in  this  way  it  is 
claimed  by  other  physiologists  who  have  repeated  the  experiments  that 
the  purines  in  the  organic  mixture  are  alone  converted,  the  non- 
nitrogenous  bodies  used  acting  merely  as  accelerators  in  the  reactions. 
The  organs  used  are  all  rich  in  the  parent  substances  of  the  purines. 

The  Formation  of  Ethereal  Sulphates.  Another  reaction  of  far- 
reaching  importance  in  the  body  is  the  production  of  organic  sulphates. 
The  oxidation  of  the  sulphur  of  proteins  leads  finally,  mainly,  to  the 
formation  of  sulphuric  acid  which  is  eliminated  in  the  urine  in  the 
form  of  the  ordinary  mineral  sulphates  and  the  ethereal  sulphates. 
The  mineral  sulphates  are  readily  formed  directly  by  combinations  in 


262  PHYSIOLOGICAL    CHEMISTRY. 

the  blood,  but  for  the  union  of  the  organic  groups  with  sulphuric  acid 
some  active  agent  is  required.  The  addition  seems  to  take  place  in 
the  liver  where  it  is  probable  that  the  oxidation  of  the  sulphur-contain- 
ing complex,  furnished  by  protein  disintegration  also  occurs.  This 
complex  seems  to  be  cystin,  C6H1204N2S2,  which  undergoes  nearly 
complete  oxidation  to  yield  sulphuric  acid  from  the  sulphur.  A  small 
portion  reaches  the  urine  finally  in  other  forms,  the  so-called  "  neutral " 
sulphur. 

Several  attempts  have  been  made  to  determine  the  seat  of  the  reac- 
tion by  irrigation  tests,  and  comparatively  recently  it  has  been  shown 
that  blood  containing  phenol  and  cystin  and  led  through  the  liver, 
freshly  dissected,  discloses  a  very  considerable  oxidation  of  the  sul- 
phur compound  with  production  of  aromatic  sulphate.  It  appears  that 
other  organs  are  not  much  concerned,  if  at  all,  in  the  reaction. 

The  aromatic  radicles  which  join  with  sulphuric  acid  in  this  way  are 
mainly  products  of  intestinal  putrefactive  changes,  and  by  absorption 
finally  reach  the  liver.  In  addition  to  sulphuric  acid  glucoronic  acid 
acts  to  hold  the  phenol  bodies ;  it  is  usually  present  in  traces  in  normal 
urine  and  is  often  greatly  increased  pathologically.  The  glucoronates 
may  be  formed  in  the  liver  along  with  the  aromatic  sulphates.  In 
experiments  which  have  been  carried  out  on  the  passage  of  the  blood 
through  a  liver  the  conjugate  phenol  bodies  produced  have  frequently 
been  in  excess  of  the  amount  called  for  by  the  sulphuric  acid  found; 
this  excess  may  correspond  in  the  main  with  the  glucoronic  acid. 

It  has  been  shown  recently  that  the  aromatic  complex  from  the  intes- 
tine and  the  sulphur  body  unite  in  the  liver  or  other  organ  only  when 
the  sulphur  group  is  not  yet  completely  oxidized.  In  other  words, 
sulphite  sulphur  and  not  sulphate  sulphur  is  here  concerned.  After 
the  union  the  final  oxidation  takes  place.  More  will  be  said  about  these 
combinations  under  the  head  of  urine  products. 

THE   BILE. 

The  formation  of  bile  is  one  of  the  important  functions  of  the  liver 
and  the  amount  secreted  in  man  is  several  hundred  grams  daily.  Some 
of  the  uses  of  the  bile  have  been  referred  to  in  earlier  chapters  under 
the  head  of  digestion  phenomena.  Other  functions  will  be  discussed 
presently. 

AMOUNT  AND  COMPOSITION. 

The  volume  of  the  bile  secreted  seems  to  be  subject  to  variations 
which  are  not  well  understood.     Through  the  aid  of  a  biliary  fistula 


THE    CHEMISTRY    OF    THE    LIVER. 


263 


it  is  possible  to  collect  the  total  excretion  in  dogs  and  other  animals 
which  are  easily  experimented  upon  and  determine  the  rate  of  flow  and 
the  whole  amount.  The  volumes  reported  by  different  observers  are 
not  in  good  agreement.  The  amounts  secreted  by  different  animals 
in  24  hours  for  each  kilogram  of  body  weight  vary  between  12  grams 
for  the  goose  and  137  grams  for  the  rabbit.  For  man  the  amounts 
observed  have  varied  between  about  150  and  1,000  grams  daily. 

The  flow  of  the  bile  is  increased,  as  far  as  volume  is  concerned  at 
any  rate,  by  the  administration  of  certain  remedies.  These  are  known 
as  cholagogues  and  among  them  calomel,  certain  resins,  rhubarb  and 
oil  of  turpentine  are  perhaps  best  known.  That  the  solids  of  the  secre- 
tion are  increased  is  a  disputed  question.  It  is  proper  to  state  here 
that  many  of  the  older  data  on  this  subject  were  obtained  by  methods 
which  are  open  to  serious  objection. 

Composition  of  Bile.  Qualitatively  bile  is  characterized  by  the 
presence  of  certain  acids  and  coloring  matters  which  are  not  found 
elsewhere  in  the  body.  The  acids  are  taurocholic  and  glycocholic,  and 
the  coloring  matters  are  bilirubin  and  biliverdin,  which  have  been 
referred  to  already  in  their  relation  to  the  coloring  matter  of  blood 
from  which  they  are  derived.  In  addition  to  these  substances  several 
others  are  present  which,  while  important,  are  not  characteristic.  These 
include  cholesterol,  fats,  soaps,  inorganic  salts  and  mucin.  The  quanti- 
tative composition  is  extremely  variable  as  shown  by  the  analyses  below, 
by  Hammarsten,  which  are  frequently  quoted.  The  results  are  in 
parts  per  1,000: 


1 

2 

3 

Water  

Solids    

974.80 
25.20 
5-29 
3-03 
6.28 
1.23 
0.63 
O.22 
0.22 
8.07 
0.25 

964.74 
35-26 
4.29 
2.08 
16.16 
1.36 
1.60 
0-57 
0.96 
6.76 
0.49 

974-60 
25.40 

Coloring  matters  and  mucin. 

5.15 
2.18 
6.86 

1. 01 

Lecithin    

1.50 
0.65 

Fat   

Soluble  salts  

0.61 

7-25 

Insoluble  salts   

0.21 

.Many  of  the  older  analyses  quoted  were  made  from  bile  from  the 
gall  bladder.  The  solids  in  the  bladder  bile  are  always  much  higher 
than  those  given  above,  because  of  a  concentration  which  takes  place 
in  that  receptable.     Some  results  for  bladder  bile  are  given  below : 


264 


PHYSIOLOGICAL    CHEMISTRY. 


Water    

Solids    

Biliary   salts    

Mucin  and  pigments 

Cholesterol    

Lecithin     

Fat    

Soaps    

Inorganic  salts   


860.0 

140.0 

72.2 

26.6 

1.6 

3-2 

£5 


859-2 

140.8 

91.4 

29.8 

2.6 

9.2 

7-7 


822.7 

177-3 

107.9 

22.1 

47-3 


10.8 


898.1 

101.9 

56.5 

14.5 

30.9 


6.2 


Analyses  have  been  made  of  bile  from  different  animals  with  the 
object  of  connecting  composition  with  the  food  of  the  animal  or  its 
habits.  The  results  are  not  very  definite.  Human  bile  contains  more 
glycocholic  than  taurocholic  acid,  while  in  carnivorous  mammals,  birds 
and  fishes  taurocholic  acid  is  the  more  abundant.  Hog  bile  contains 
largely  glycocholic  acid,  but  in  ox  bile  the  relation  is  variable.  The 
amounts  of  the  pigments  are  small  and  not  accurately  known. 

Glycocholic  Acid.  This  is  a  complex  substance  made  up  of  a  com- 
bination of  glycocoll  or  glycine  with  cholalic  acid.  The  constitution 
of  the  acid  is  not  known,  but  the  empirical  formula  C26H43N06  has 
been  given  to  it.  In  the  bile  it  exists  in  the  form  of  a  sodium  or 
potassium  salt,  which  is  readily  soluble  in  water  or  alcohol.  The  free 
acid  is  but  slightly  soluble ;  hence  the  addition  of  mineral  acids  to  bile 
produces  a  precipitate.  On  boiling  a  solution  of  glycocholic  acid  with 
weak  acids  or  alkalies  a  cleavage  follows,  and  glycocoll  and  the  nitrogen- 
free  cholalic  acid  separate.  Water  is  taken  up  at  the  same  time.  This 
is  a  reaction  analogous  to  the  separation  of  glycocoll  and  benzoic  acid 
from  hippuric  acid  by  the  same  manner  of  treatment.  There  appear 
to  be  several  cholalic  acids,  but  with  the  common  one  the  reaction 
would  be  represented,  probably,  in  this  way : 

C6H43NOe  +  H20  =  C2H302NH2  +  C24H4o06. 


Taurocholic  Acid.  To  this  substance  the  empirical  formula 
C26H45NS07  is  given.  With  weak  acids  it  undergoes  likewise  a 
hydrolytic  cleavage  from  which  taurin  and  cholalic  acid  result.  Taurin 
appears  to  be  aminoethylsulphonic  acid,  C2H4NH2-HS03,  and  the 
cleavage  would  be  represented  in  this  way : 

C26H«NS07  +  H20  =  C24H4»05  +  C,ILNH2HS03. 

The  free  acid  has  a  bitter-sweet  taste ;  it  is  much  more  soluble  in  water 
than  the  glycocholic  acid  and  somewhat  soluble  in  alcohol.  The  free 
acid  has  the  property  of  holding  glycocholic  acid  in  aqueous  solution, 
which  is  shown  by  the  difficulty  in  precipitating  the  mixed  acids  from 


THE    CHEMISTRY    OF    THE    LIVER.  265 

ox  bile.  The  free  acid  is  but  slightly  soluble  in  ether.  The  alkali  salts 
are  soluble  in  water  and  alcohol. 

Cholalic  Acid.  Although  many  investigations  have  been  carried 
out  with  this  substance  its  constitution  is  not  clear.  The  above  em- 
pirical formula,  C24H40O5,  is  that  of  a  monobasic  acid  to  which  Mylius 
has  given  this  possible  structure, 

fCHOH 

c»n»1  CH2OH 
[cOOH 

The  free  acid  is  very  slightly  soluble  in  water,  but  the  alkali  salts  are 
readily  soluble.  The  free  acid  is  somewhat  soluble  in  ether;  hence  it 
is  found  as  a  decomposition  product  of  the  bile  acids  in  the  crude  fat 
extracted  from  feces.  By  oxidation  cholalic  acid  yields  several  new 
acids  which  have  been  much  studied  with  the  hope  of  gaining  an  insight 
into  the  structure  of  the  original  acid.  Among  the  various  derived 
acids  these  may  be  mentioned :  Choleic  acid,  C25H4204,  dehydrocholeic 
acid,  C24H3404,  cholanic  acid,  C24H34Os.  Fellic  acid,  C23H40O4,  and 
lithofellic  acid,  C20H36O4,  are  found  in  some  kinds  of  bile. 

Preparation  of  Acids  from  Ox  Bile.  This  may  be  illustrated  by  the  following. 
Evaporate  200  to  300  cc.  of  the  bile  to  dryness,  or  as  near  to  dryness  as  possible,  on 
the  water-bath  with  the  addition  of  about  60  grams  of  bone-black.  After  cooling 
the  mass  rub  it  up  thoroughly,  transfer  to  a  flask  and  extract  with  alcohol  by 
heating  over  a  water-bath.  The  two  bile  salts  are  soluble  in  the  alcohol,  while 
the  mucin  and  inorganic  salts  present  are  not.  Therefore  cool  the  extracted  mix- 
ture and  filter.  The  filtrate  contains  the  bile  salts  along  with  cholesterol,  some  fat 
and  traces  of  other  substances.  There  is  also  some  water  present.  Evaporate  the 
filtrate  to  dryness,  take  up  with  absolute  alcohol  and  filter  again.  Taking  advan- 
tage of  the  practical  insolubility  of  the  bile  salts  in  ether  they  may  be  precipitated 
in  this  way :  Add  to  the  strong  alcoholic  solution  an  excess  of  dry  ether,  or  enough 
to  cloud  the  mixture,  and  allow  to  stand.  After  some  hours  or  days  a  crystalline 
precipitate  of  the  bile  salts  separates.  The  crystals  may  be  used  for  preparation  of 
other  substances,  or  for  tests.  In  the  mother  liquor  cholesterol  may  be  detected  by 
the  tests  given  in  an  earlier  part  of  this  work. 

Preparation  of  Glycocholic  Acid.  Use  the  larger  part  of  the  above  described 
crystalline  precipitate  for  this  purpose.  Dissolve  in  water  and  add  enough  dilute 
sulphuric  acid  to  produce  a  marked  turbidity.  Add  a  little  ether,  shake  the  mix- 
ture well  and  allow  to  stand  in  a  cold  place.  The  glycocholic  acid  separates  in  the 
form  of  fine  silky  needles.  Press  out  the  mother  liquor,  redissolve  in  hot  water 
and  allow  to  crystallize  a  second  time.     A  nearly  pure  product  may  be  so  obtained. 

Preparation  of  Taurocholic  Acid.  The  separation  of  this  acid  from  the  glyco- 
cholic acid  is  extremely  difficult,  hence  in  preparing  it,  it  is  best  to  start  with  a  bile 
which  contains  essentially  only  the  one  salt.  Dog's  bile  should  therefore  be  em- 
ployed. Treat  it  as  described  for  the  mixed  salts,  and  decompose  finally  with  dilute 
sulphuric  acid  in  presence  of  ether. 

Preparation  of  Cholalic  Acid.  Dissolve  200  grams  of  barium  hydroxide  in  6 
liters  of  water.     In  this  solution  saponify  50  gm.  of  glycocholic  acid,  by  boiling 


266 


PHYSIOLOGICAL    CHEMISTRY. 


ten  to  twenty  hours,  replacing  the  water  lost  by  evaporation.  Filter  hot  and  to 
the  cooled  liquid  add  enough  hydrochloric  acid  to  decompose  the  barium  salt. 
The  cholalic  acid  separates  in  the  form  of  a  granular  precipitate.  Wash  with  water 
and  crystallize  from  hot,  strong  alcohol. 

Optical  Properties  of  These  Acids.  The  three  acids  and  their  sodium  salts 
are  characterized  by  rather  strong  rotating  power,  which  under  some  circumstances 
may  be  used  for  measurement  or  identification.  The  following  specific  rotations 
have  been  found : 


For  Aqueous  Solution. 

For  Alcohol  Solution. 

c 

Wj, 

c 

Wa 

Taurocholic  acid,  sodium  salt 

Cholalic  acid,  anhydrous  

24.928 
8.856 

I9.O49 

+20.80 
+  21.5 

+26.0 

9-5°4 
20. 143 
9.898 
2.942 
2.230 

-I-29.00 

+  25.7 

424.5 
447.6 

431-4 

Chemical  Test  for  the  Bile  Salts.  The  three  acids  are  characterized  by  giving 
a  certain  reaction  with  furfuraldehyde,  or  sugar  yielding  furfuraldehyde,  in  presence 
of  acid.  The  test  is  commonly  made  by  adding  to  a  dilute  solution  of  the  salts, 
say  5  cubic  centimeters,  a  few  drops  of  a  dilute  cane  sugar  solution  and  strong 
sulphuric  acid  in  volume  about  half  that  of  the  mixture.  Let  the  acid  flow  down 
the  side  of  the  test-tube  so  as  to  form  a  layer  below  the  lighter  liquid.  A  deep 
purple  band  appears  at  the  line  of  contact.  On  slowly  mixing  the  liquids  in  the 
test-tube  the  color  becomes  purple  throughout.  In  this  test  any  excess  of  sugar  must 
be  avoided. 

With  a  trace  of  pure  furfuraldehyde  in  place  of  sugar  the  reaction  is  sharper, 
but  certain  proportions  must  be  observed.  A  good  mixture  is  1  cubic  centimeter 
of  weak  alcoholic  solution  of  the  bile  acid,  1  drop  of  0.1  per  cent  furfuraldehyde 
solution  and  1  cubic  centimeter  of  strong  sulphuric  acid.  The  original  test  was 
devised  by  Pettenkofer;  later  it  was  recognized  that  the  reaction  belongs  to  the 
group  of  "  f  urf  urol "  reactions,  and  the  aldehyde  was  recommended  in  place  of 
the  sugar.  The  test  cannot  be  used  with  bile  directly  because  of  the  presence  of 
other  substances,  which  would  give  a  strong  color  with  the  sulphuric  acid. 

Preparation  of  Taurin.  Use  several  hundred  cubic  centimeters  of  ox  bile.  Add 
to  it  an  excess  of  strong  hydrochloric  acid,  about  one-third  of  the  volume  of  the 
bile,  and  boil  on  the  water-bath.  A  resinous  mass  separates  and  when  this  be- 
comes stringy  enough  to  solidify,  when  a  little  is  taken  up  on  a  rod  and  allowed  to 
cool,  the  reaction  has  gone  far  enough.  Decant  from  this  mass  and  evaporate  the 
liquid  resulting  until  a  crystallization  of  salt  forms.  Filter  and  evaporate  to  a 
small  volume.  If  salt  separates  filter  again  and  pour  the  liquid  finally  into  a  large 
excess  of  alcohol.  This  causes  the  taurin  to  separate;  wash  the  crude  substance 
with  strong  alcohol,  and  recrystallize  from  hot  water.  In  a  successful  separa- 
tion large  plates  or  prisms  of  taurin  are  obtained.  The  substance  may  be  recog- 
nized by  several  tests.  On  heating  it  chars  and  gives  off  an  odor  of  sulphurous 
acid.  When  fused  with  sodium  carbonate  the  sulphur  is  converted  into  sulphide, 
from  which  hydrogen  sulphide  may  be  separated  and  identified  by  the  usual  tests. 

THE  BILE  PIGMENTS. 

The  two  substances,  biliverdin  and  bilirubin,  are  related  to  hematin 
from  hemoglobin,  as  pointed  out  above,  and  as  may  be  illustrated  by 
these  formulas : 


THE    CHEMISTRY    OF    THE    LIVER.  267 

Hematin    C32H32N404Fe 

Hematoporphyrin    C16HiSN203,  or  C32H3cN406 

Bilirubin    C16H1SN203,  or  C32H3eN406 

Biliverdin    C]6H18N204,  or  C32H36N408 

The  two  bile  pigments  are  formed  in  the  liver  and  normally,  appar- 
ently, only  in  the  liver,  but  by  what  kind  of  reaction  is  not  clearly 
known.  Hematoporphyrin  may  be  produced  from  hematin  and  it  is 
isomeric  with  bilirubin,  though  not  identical.  The  relation  of  bilirubin 
to  blood  is  perhaps  best  shown  by  this  observation :  in  old  blood  extrav- 
asations the  blood  color  appears  to  be  gradually  decomposed  and  in  its 
place  the  new  coloring  matter  is  found,  which  was  called  hematoidin 
by  its  discoverer.  Later  studies  have  apparently  shown  the  identity 
of  this  with  bilirubin. 

Bilirubin  is  practically  insoluble  in  water,  but  it  seems  to  act  as  an 
acid,  the  alkali  salts  of  which  are  soluble.  In  this  form  it  exists  in  bile. 
The  solution  is  reddish  yellow  and  in  the  air,  or  by  treatment  with 
oxidizing  agents,  it  takes  up  oxygen  and  becomes  biliverdin,  which 
gives  a  green  solution.  The  bile  always  contains  the  two  pigments, 
from  which  the  greenish  yellow  color  follows.  The  amount  of  the 
two  substances  in  the  bile  is  normally  very  small,  but  as  the  reactions 
are  sharp  recognition  is  easy.  The  total  weight  of  the  two  pigments 
produced  in  one  day  is  not  over  200  milligrams  probably;  the  physio- 
logical meaning  of  the  formation  is  not  known.  The  iron  of  the  orig- 
inal hematin  is  largely  retained  by  the  substance  of  the  liver  cells. 

Preparation  of  Bilirubin.  The  pigment  cannot  be  easily  obtained  from  bile 
because  of  the  small  amount  present,  but  may  be  obtained  from  the  pathological 
concretions  known  as  gall-stones,  which  will  be  described  later.  Powder  several 
grams  of  these  stones  from  cattle  very  fine  and  exhaust  thoroughly  with  ether,  then 
repeatedly  with  boiling  water  to  take  out  cholesterol  and  bile  acids.  In  the  residue 
the  bilirubin  exists  as  an  insoluble  calcium  compound ;  this  is  decomposed  by  the 
addition  of  a  little  dilute  hydrochloric  acid,  after  which  what  is  left  is  washed 
thoroughly  with  hot  water,  and  then  with  alcohol  to  leave  the  pigment  in  a  still  better 
condition  for  extraction.  Finally  extract  with  chloroform  in  which  the  substance 
is  relatively  soluble.  On  evaporating  the  chloroform  crude  bilirubin  is  secured, 
which  after  washing  with  alcohol  may  be  recrystallized  from  hot  chloroform  or 
from  dimethylaniline,  in  which  it  dissolves  in  the  proportion  of  about  1  to  100 
cold  or  1  to  30  hot.  By  several  crystallizations  it  is  possible  to  obtain  a  product 
pure  enough  to  employ  as  a  standard  for  spectroscopic  measurements. 

I'.y  exposing  an  alkaline  solution  to  the  air  or  by  treating  with  a  little  acid  and 
sodium  peroxide,  bilirubin  is  converted  into  biliverdin.  The  latter  free  substance 
is  not  soluble  in  water,  chloroform  or  ether. 

The  Bile  Pigment  Tests.  Some  of  these  are  extremely  delicate  and  have  long 
been  used  for  the  recognition  of  bile,  especially  in  urine.  For  the  test  to  be 
givf-n  the  bilirubin  alkali  in  very  dilute  solution  may  be  used,  or  a  diluted  bile. 

i.in's  Test.     In  a  test-tube  take  a  few  cubic  centimeters  of  nitric  acid  con- 
taining some  nitrous  acid.     Over  this  pour  carefully  the  weak  bile  solution  to  be 


268  PHYSIOLOGICAL    CHEMISTRY. 

tested.  At  the  junction  point  colored  rings  appear  which  result  from  the  forma- 
tion of  oxidation  products  of  the  bilirubin.  The  colors  appear  in  this  order  from 
above  down :  green,  blue,  violet,  red  and  yellowish.  Of  these  the  green  is  the  most 
characteristic;  the  other  shades  represent  more  advanced  stages  in  the  oxidation. 
For  success  in  the  test  the  bile  solution  must  not  be  too  strong,  and  the  amount  of 
nitrous  acid  in  the  nitric  acid  must  be  small. 

Hammarsten's  Test.  Use  as  reagent  a  mixture  of  strong  nitric  acid  and  strong 
hydrochloric  acid  in  the  proportion  of  about  I  to  50  by  volume.  This  mixture  must 
stand  some  time  before  use,  or  until  it  becomes  yellow.  It  keeps  a  long  time.  For 
the  practical  test  mix  i  cubic  centimeter  of  the  acid  with  4  cubic  centimeters  of 
alcohol  and  add  a  drop  or  two  of  the  bilirubin  solution  to  be  tested.  A  perma- 
nent green  color  appears,  but  if  strong  oxidation  is  secured  by  adding  more  of  the 
acid  mixture  the  colors  change  as  in  the  Gmelin  test.  The  reaction  can  be  well 
applied  to  urine. 

FUNCTIONS  AND  BEHAVIOR  OF  BILE. 

The  bile  as  a  whole  has  a  number  of  functions  to  perform  in  the 
body,  some  of  which  have  been  referred  to  in  the  discussion  of  diges- 
tive processes.  It  represents  also  the  avenue  of  escape  of  a  number 
of  by-products  formed  by  the  katabolic  processes  in  the  liver.  Many 
of  these  processes  are  doubtless  very  complex  and  in  them  a  variety  of 
secondary  or  side  reactions  occur  which  furnish  matters  of  no  further 
use  apparently  in  the  body.  These  are  collected  in  the  gall  bladder 
and  finally  discharged  into  the  small  intestine,  where  escape  from  the 
body  with  the  feces  is  possible  for  the  constituents  having  no  further 
value.  If  the  escape  of  these  products  from  the  liver  is  hindered,  some 
form  of  icterus  results,  as  the  bodies  in  question  must  pass  more  or 
less  directly  into  the  blood. 

In  part,  therefore,  the  bile  must  be  regarded  as  an  excretion  like 
the  urine,  but  that  the  parallelism  is  not  complete  is  shown  by  the  fact 
that  a  considerable  absorption  takes  place  from  the  intestine,  and 
products  are  returned  which  find  further  application  in  the  organism. 
There  is  evidence  to  show  that  this  portion  returned  from  the  intestine 
serves  as  a  cholagogue  to  stimulate  new  secretion  in  the  liver.  It  is 
likely  that  this  free  secretion  and  flow  of  bile  in  the  liver  is  necessary 
for  the  successful  completion  of  certain  metabolic  processes  going  on 
there,  so  that  it  may  be  regarded  not  merely  as  an  end  but  also  as  a 
means  toward  an  end. 

The  one  digestive  process  in  which  the  bile  seems  to  play  a  practically 
necessary  part  is  in  the  splitting  and  absorption  of  fats ;  here  its  action 
is  partly  mechanical  as  in  some  way  it  aids  the  passage  of  the  finely 
divided  fat  through  the  intestinal  walls.  The  general  behavior  of  bile 
in  this  respect  may  be  illustrated  by  a  simple  experiment. 


THE    CHEMISTRY    OF   THE   LIVER.  269 

Experiment.  Moisten  two  similar  filter  papers  in  funnels,  one  with  water  and 
the  other  with  bile.  Into  each  filter  pour  some  fatty  oil,  such  as  cotton-seed  oil 
or  olive  oil.  Note  that  while  the  oil  will  not  pass  through  the  paper  moistened 
with  water  a  small  amount  passes  slowly  through  the  bile-moistened  filter.  Similar 
experiments  have  been  made  with  animal  membranes. 

A  more  important  action  with  fat,  however,  is  shown  in  the  power 
of  bile  to  form  fat  emulsions,  which  depends  on  the  behavior  of  the 
bile  salts  with  the  fat  splitting  ferment,  as  already  pointed  out,  and  on 
the  formation  of  soaps  directly.  This  is  now  looked  upon  as  the  one 
reaction  in  the  intestine  in  which  the  presence  of  bile  is  actually  prac- 
tically essential,  since  the  old  views  of  the  antiseptic  value  of  the  bile 
in  preventing  excessive  intestinal  putrefaction  have  been  shown  to  be 
without  foundation.  In  a  diet  rich  in  fats  the  emulsifying  behavior 
of  the  bile  unquestionably  comes  into  play  as  a  leading  factor  in  the 
final  absorption.  Experiments  have  been  made  on  animals  in  which 
the  flow  of  the  bile  could  be  diverted  from  the  natural  outlet  into  the 
intestine  by  means  of  a  fistula.  In  such  cases  the  digestion  of  proteins 
and  carbohydrates  seemed  to  suffer  no  change  but  the  digestion  of  fats 
was  always  imperfect  and  a  large  portion  ultimately  escaped  with  the 
feces.  Indirectly  there  may  be  also  a  loss  in  protein  if  the  fat  in  the 
food  in  such  cases  has  a  rather  low  melting  point  and  is  abundant.  A 
fatty  layer  encloses  portions  of  the  partly  digested  proteins  and  pre- 
vents access  of  the  digestive  fluids  until  the  lower  stretches  of  the  intes- 
tine are  reached,  where  bacterial  changes  soon  get  the  upper  hand  and 
rob  the  protein  of  any  further  food  value.  The  action  of  bile  in  pro- 
ducing an  emulsion  with  fatty  oils  may  be  illustrated  by  experiment. 
In  an  earlier  chapter  the  formation  of  emulsions  by  other  methods  was 
shown. 

Experiment.  In  a  slightly  warmed  mortar  pour  about  5  cc.  of  bile  and  add  to 
it  one  cc.  of  cottonseed  oil.  Rub  the  two  thoroughly  together  for  several  minutes, 
and  then  add  another  small  portion  of  the  fatty  oil.  An  emulsion  forms  slowly, 
and  becomes  more  persistent  as  the  working  with  the  pestle  is  prolonged.  The 
amount  of  oil  which  can  be  brought  into  the  form  of  a  stable  emulsion  with  the 
5  cc.  of  bile  depends  largely  on  the  character  of  the  oil.  The  presence  of  a  small 
amount  of  free  fatty  acid  in  the  cottonseed  oil  aids  materially  in  producing  the 
emulsion.  The  weak  alkalinity  of  the  bile  is  doubtless  an  important  point  here,  as 
through  the  alkali  a  little  soap  is  formed  and  this  may  be  the  chief  factor  in  pro- 
ducing the  emulsion. 

In  the  intestines  the  stimulating  action  of  the  bile  salts  is  probably 
more  important  than  this  last  reaction.  At  the  present  time  these  salts 
are  prepared  in  comparatively  pure  form  as  medicinal  agents. 

Bile  contains  a  large  amount  of  mucin  as  the  analytical  table  above 
shows.     The  stringy  character  of  the  secretion  is  due  to  this  substance 


270  PHYSIOLOGICAL    CHEMISTRY. 

which  may  be  recognized  by  several  precipitation  tests.  The  addition 
of  alcohol  in  excess  throws  down  a  flocculent  mass  which  may  be  sepa- 
rated by  the  centrifuge.  The  addition  of  a  little  acetic  acid  produces 
likewise  a  precipitate.  It  is,  however,  practically  impossible  to  secure 
pure  mucin  in  this  way  as  other  bodies  are  carried  down  with  the  pre- 
cipitates and  their  subsequent  separation  is  difficult.  The  mucin  of 
human  bile  is  said  to  be  nearly  pure,  while  that  of  other  animals  is 
mixed  with  nucleo  albumins. 

BILE  CONCRETIONS.     GALL  STONES. 

Under  conditions  not  well  understood  a  precipitation  of  certain  con- 
stituents of  the  bile  may  occur  in  the  gall  bladder.  These  precipita- 
tions take  the  form  of  solid  masses  which  sometimes  grow  to  consid- 
erable size,  by  gradual  surface  additions.  In  every  case  the  deposited 
material  is  built  up  in  layers,  often  well  defined,  around  some  body  as 
a  nucleus.  Three  general  classes  of  such  calculi  are  recognized.  In 
man  balls  of  cholesterol,  more  or  less  pure,  are  the  most  abundant  while 
pigment  stones  are  also  frequently  found.  These  pigment  stones  con- 
tain essentially  bilirubin  in  combination  with  calcium,  the  alkali  earth 
salts  of  the  pigments  being  insoluble.  The  center  of  the  cholesterol 
stone  may  be  a  nucleus  of  bilirubin  calcium.  Pigment  stones  are  com- 
mon in  the  gall  bladders  of  cattle.  Finally  we  have  stones  consisting 
of  calcium  phosphate  or  carbonate,  which,  however,  are  not  usual  in 
man. 

The  following  analyses  made  of  gall-stones  of  very  different  appear- 
ance illustrate  the  composition  of  the  cholesterol  stones  in  man : 

Water  (at  ioo°)   4.60  4.50 

Cholesterol  (and  trace  of  fat) 90.87  90.08 

Bilirubin  (CHC13  extraction) 0.81  ~l  H                 0.19 ~j 

Biliverdin  (C2H,0  extraction)    2.24  J3-05  1.58  J  I-77 

Mucin  and  soluble  extractives    0.14  1.53 

Total  ash   0.88  2.72 

Total  P205   0.20  1.00 

These  concretions  frequently  give  rise  to  serious  pathological  condi- 
tions and  they  must  then  be  removed  by  surgical  operations.  In  addi- 
tion to  the  above  constituents  the  stones  contain  small  amounts  of  iron 
and  often  traces  of  copper.  But  the  iron  found  is  far  from  accounting 
for  the  amount  which  must  be  separated  from  the  hematin  in  the  for- 
mation of  bilirubin.  In  a  former  chapter  the  preparation  of  choles- 
terol from  gall-stones  was  described,  also  the  general  chemical  behavior 
of  the  substance.     The  character  of  a  stone  is  most  easily  recognized 


THE    CHEMISTRY    OF   THE    LIVER.  27 1 

by  its  behavior  toward  boiling  alcohol,  in  which  cholesterol  is  rather 
readily  soluble,  to  crystallize  in  large  thin  plates  on  cooling. 

The  solutions  of  cholesterol  have  a  marked  action  on  polarized  light, 
which  property  may  be  employed  sometimes  in  the  identification  and 
estimation.     The  specific  rotations  below  have  been  found. 

Ether  solution  c  =  2  [a]ols  =  —  31.12° 

Chloroform  solution  "      2    "  —  37-02° 

"      5    "  -  37.8i  ° 

"      8    "  -38-63° 

In  feces  a  modified  cholesterol  is  found  which  has  been  called  koprosterin  and  also 
stercorin.  This  new  substance  is  a  reduction  product  with  the  probable  formula 
QrH^O  and  is  dextrorotatory,  [a]  =  -f  24°. 

Besides  the  two  principal  pigments  several  derived  substances  have  been  obtained 
from  the  gall-stones.  The  following  have  been  described:  bilifuscin,  biliprasin,  bill- 
hum  in,  bilicyanin.  These  substances  exist  in  small  amount  and  are  without  practical 
importance.     Their  relations  to  the  others  are  not  clearly  established. 


CHAPTER   XVIII. 

CHEMISTRY  OF  THE  PANCREAS  AND  OTHER  GLANDS.     MUSCLE, 
BONE,  THE  HAIR  AND  OTHER  TISSUES. 

In  this  chapter  a  number  of  substances  will  be  briefly  discussed,  the 
chemical  relations  of  which  in  some  cases  are  unimportant,  or  some- 
times, when  important,  not  well  understood.  In  regard  to  the  pan- 
creas, it  will  be  recalled  that  in  the  discussion  of  digestive  phenomena 
the  behavior  of  active  enzymes  in  the  liquid  secreted  by  the  organ  was 
rather  fully  considered.  In  the  so-called  pancreatic  juice  the  three 
most  important  enzymes  are  active  in  the  digestion  of  carbohydrates, 
fats  and  proteins,  but  in  addition  to  these  functions  others  must  be 
mentioned. 

THE   PANCREAS. 

The  organ  is  relatively  poor  in  solids,  containing  only  about  ioo 
parts  per  1,000.  The  solid  substance  consists  largely  of  nucleo-proteids 
with  but  comparatively  small  amounts  of  the  other  protein  bodies. 
Besides  producing  the  digestive  enzymes,  or  their  zymogens,  the  pan- 
creas cells  have  an  important  function  to  perform  in  connection  with 
the  oxidation  of  sugar  in  the  body.  It  has  long  been  known  that  a 
kind  of  diabetes  results  on  the  extirpation  of  the  pancreas.  Something 
seems  to  be  produced  there  which  is  apparently  essential  in  the  oxida- 
tion process.  Experiments  with  animals  have  shown  that  the  oxida- 
tion takes  place  if  even  a  small  portion  of  the  organ  is  left.  Of  the 
nature  of  the  active  ferment  here  or  of  its  mode  of  action  practically 
nothing  is  known;  but  it  has  been  pointed  out  recently  by  several 
writers  that  in  this  sugar  oxidation,  taking  place  in  the  muscles  prob- 
ably, two  things  are  concerned.  The  pancreas  may  furnish  one  of 
these  and  an  enzyme  formed  in  the  muscle  cells  is  the  other.  Cell-free 
extracts  from  the  organs  taken  separately  have  been  found  to  be  prac- 
tically inert  toward  sugar,  while  in  presence  of  a  mixture  of  the  two 
extracts  oxidation  follows  readily.  It  has*  been  suggested  that  one  of 
these  organs  furnishes  an  enzyme  which  is  the  catalyzer  for  the  other, 
and  attempts  have  been  recently  made  to  produce  the  pancreas  enzyme 
on  a  large  scale  for  use  therapeutically. 

Autolysis.  The  pancreas  readily  undergoes  autolytic  digestion 
under  the  aseptic  treatment  or  when  preserved  by  toluene.     A  large 

272 


CHEMISTRY  OF  THE   PANCREAS   AND   OTHER   GLANDS.  273 

number  of  products  may  be  separated  from  the  altered  mass,  which  in 
a  general  way  resemble  those  produced  in  the  liver,  as  already  referred 
to.  Ammonia,  leucine,  tyrosine,  aspartic  acid,  glutaminic  acid  and  the 
hexone  bases  have  been  recognized;  also,  the  somewhat  unusual  oxy- 
phenylethylamine,  HO-C6H4-CH2-CH2NH2,  which  may  be  derived 
from  tyrosine  by  splitting  off  of  C02. 

On  account  of  the  relatively  high  content  of  nucleo-proteids,  and  the 
constituent  nucleic  acids,  a  marked  amount  of  sugar  in  the  form  of  pen- 
tose is  liberated.  No  other  organ  subjected  to  prolonged  autolysis 
seems  to  yield  as  much.  In  certain  pathological  conditions  involving 
the  pancreas,  the  urine  contains  a  complex  which  yields  a  pentose  on 
treatment  with  acid  at  the  boiling  temperature.  The  pentose  is  iden- 
tified through  its  phenyl  hydrazine  compounds. 

THE   SUPRARENAL   BODIES. 

A  soluble  substance  contained  in  the  capsules,  because  of  its  impor- 
tant property  of  raising  the  blood  pressure,  has  attracted  a  great  deal 
of  attention  in  the  last  ten  years.  This  soluble  substance  was  first 
recognized  as  a  chromogen  which,  on  account  of  its  oxygen-absorbing 
power,  was  assumed  to  be  related  to  pyrocatechol.  An  aqueous  extract 
of  the  capsules  becomes  dark  on  exposure  to  the  air  and  produces  a 
dark  green  color  when  treated  with  ferric  chloride.  It  also  reduces 
Fehling's  solution  strongly  and  shows  the  same  behavior  toward  other 
metallic  salts.  The  oxygen-absorbing  power  of  the  extract  had  been 
known  about  thirty  years  before  the  important  relation  to  blood  pres- 
sure was  discovered.  It  was  soon  found  that  the  two  properties  seem 
to  reside  in  the  same  constituent  of  the  extract,  since  the  destruction  of 
one  is  followed  by  the  disappearance  of  the  other.  Numerous  investi- 
gations have  been  carried  out  on  the  isolation  of  the  active  principle, 
especially  by  Abel,  v.  Fiirth  and  Takamine,  who  have  given  the  names 
epinephrin,  suprarenin  and  adrenalin  respectively  to  active  extracts 
which  they  have  separated  by  different  processes.  Some  idea  of  the 
nature  of  the  substance  may  be  obtained  from  considering  a  method 
given  by  Takamine  for  separating  it. 

The  minced  capsules  are  extracted  by  weakly  acidulated  water  in  an  atmosphere 
of  carbon  dioxide  to  prevent  oxidation.  The  temperature  of  the  extraction  is  at 
first  5o°-6o°  and  finally  90°-95°  to  coagulate  proteins.  The  extract  is  con- 
centrated in  vacuo  and  precipitated  with  strong  alcohol;  the  filtrate  is  concen- 
trated— the  alcohol  distilled  off — in  vacuo,  and  to  the  aqueous  residue  ammonia  is 
added.  This  produces  a  precipitate  of  the  active  principle  in  crude  form,  which  crys- 
tallizes in  time.  The  precipitate  is  redissolved  with  a  little  acid  in  alcohol,  and 
certain   impurities  are   thrown   out  by  addition   of   ether.     The   filtrate   is   concen- 

19 


274  PHYSIOLOGICAL    CHEMISTRY. 

trated  in  vacuo  again  and  a  new  precipitation  effected  by  ammonia.     By  repeating 
this  treatment  several  times  a  much  purer  product  is  obtained. 

A  light  yellowish  crystalline  powder  is  secured,  which  is  somewhat 
soluble  in  water.  It  combines  with  hydrochloric  acid  to  form  the  stable 
salt  commonly  used  in  medicine.  The  empirical  formula  is  C9H13N03 
and  for  this  several  constitutional  formulas  have  been  suggested. 

The  other  constituents  of  the  suprarenal  capsules  have  no  impor- 
tance at  the  present  time  that  can  be  clearly  denned,  but  as  is  well 
known,  complete  removal  of  the  bodies  is  usually  attended  with  fatal 
results,  and  Addison's  disease  is  associated  with  certain  pathological 
conditions  in  the  organs.  Lecithin  bodies  and  a  glucose-furnishing 
complex  are  present  in  small  amount,  as  well  as  the  mass  of  protein 
substance  which  has  not  yet  been  fully  investigated. 

THE   THYROID    GLAND. 

The  relation  of  this  gland  to  certain  pathological  conditions  which 
sometimes  appear  in  man  and  which  may  be  induced  in  animals  has 
been  a  subject  of  study  for  many  years.  Attempts  to  isolate  the  active 
principle  or  principles  on  which  the  functions  of  the  gland  depend  have 
been  in  a  measure  successful.  In  the  course  of  investigations  a  number 
of  basic  bodies  have  been  separated,  but  these  may  have  no  connection 
with  the  observed  physiological  behavior. 

From  the  investigations  of  Oswald,  who  has  made  the  fullest  con- 
tributions to  the  literature,  there  are  two  peculiar  protein  bodies  present, 
one  of  which  is  a  globulin  and  the  other  a  nucleo-proteid.  To  the  first 
he  has  given  the  name  thyreo-globulin;  this  exists  frequently  combined 
with  iodine,  and  it  is  the  latter  complex  which  is  assumed  to  be  theo- 
retically and  practically  important.  It  has  been  called  iodothyreo- 
globulin  and  appears  to  be  found  only  in  those  glands  which  contain 
colloid,  and  the  amount  of  iodine  present  is  proportional  to  the  amount 
of  colloid.  The  normal  gland  weighs  usually  30-45  grams,  in  which 
the  thyreoglobulin  fraction  is  in  the  mean  about  ten  per  cent.  The 
amount  of  iodine  is  usually  less  than  one  tenth  of  one  per  cent  of  the 
whole.  In  case  of  enlarged  glands — goitre — the  whole  organ  may 
weigh  up  to  several  hundred  grams.  If  the  goitre  is  rich  in  colloid 
the  iodine  appears  to  be  absolutely,  but  not  relatively,  increased.  In 
the  thyreo-globulin  from  a  normal  gland  over  0.3  per  cent  of  iodine 
has  been  found,  while  in  the  preparation  from  colloid  goitres  the 
amount  in  the  mean  is  0.06-0.07  Per  cent. 

By  treatment  with  acids  the  gland,  or  the  thyreo-globulin  from  it, 
undergoes  a  cleavage  in  which  a  residue  rich  in  iodine  remains.     The 


CHEMISTRY  OF  THE   PANCREAS  AND   OTHER  GLANDS.  2J5 

organic  iodine  compound  so  obtained  which  may  be  the  true  active 
principle  is  called  iodothyrin  or  thyroiodine.  In  earlier  experiments 
Baumann,  the  discoverer  of  this  compound,  found  an  iodine  content  of 
about  9  per  cent,  but  Oswald,  starting  with  pure  iodothyreo-globulin 
which  was  secured  by  a  salting-out  process  with  ammonium  sulphate, 
obtained  finally  iodothyrin  with  over  14  per  cent  of  combined  iodine. 
This  iodothyrin  is  not  a  protein  substance;  the  analyses  of  different 
preparations  are  not  in  very  good  accord,  from  which  it  appears  that 
the  pure  substance  has  not  yet  been  actually  secured.  The  crude 
product  at  present  known  has  been  used  in  medicine  and  attempts  have 
been  made  to  duplicate  or  replace  it  by  other  iodine  compounds. 

It  is  now  generally  recognized  that  the  physiological  activity  of  the 
dried  thyroid  on  the  market  in  powdered  form  is  proportional  to  the 
iodine  content.     No  exact  method  of  valuation  is  known. 

The  smaller  glands  associated  with  the  thyroid  and  known  as  the 
parathyroids  are  possibly  even  more  important.  Both  sets  of  glands 
have  apparently  much  to  do  with  the  general  metabolic  functions  of  the 
body,  and  the  complete  removal  of  the  parathyroids  is  usually  followed 
by  death.     How  they  act  is  not  clearly  known. 

THE   REPRODUCTIVE    GLANDS. 

Of  the  chemical  composition  of  the  testicles  and  their  secretion  not 
much  can  be  said.  The  testicles  contain  several  proteins  and  extrac- 
tives, but  their  investigation  has  been  extremely  limited.  The  most 
complete  examinations  of  the  spermatic  fluid  are  probably  those  re- 
ported by  Slowtzoff,  from  whose  work  the  following  figures  are  taken. 
The  specific  gravity  of  the  fluid  varies  from  1.02  to  1.04;  the  reaction 
is  alkaline  and  as  measured  by  the  aid  of  rosolic  acid  corresponds  to 
0.15  per  cent  sodium  hydroxide.  As  a  mean  of  five  analyses  the  fol- 
lowing results  may  be  given : 

Spermatic  Fluid. 

Specific  gravity 1.0299 

Water    90.32  per  cent. 

Dry  substance  9.68 

Salts    0.90 

Proteins    2.09 

Ether  extract   0.17 

Water  and  alcohol  extracts   6.1 1 

The  tables  below  show  the  calculations  for  dry  substance  and  the 
character  of  the  ash  : 


276  PHYSIOLOGICAL    CHEMISTRY. 

For  Dry  Substance.  Ash. 

Organic    90.81  per  cent.        NaCl    29.05  per  cent. 

Inorganic   9.19  "  KC1    3-12 

Proteins   24.48  "  S03   11.72 

Ether  extract  2.15  "  CaO  22.40         " 

Water  and  alcohol  ex-  P205    28.79 

tract    59.36  " 

The  ash  is  peculiar  in  containing  a  large  amount  of  sodium  chloride 
and  calcium  phosphate.  The  phosphoric  acid  is  present  in  larger 
amount  than  corresponds  to  the  nuclein  substances. 

The  proteins  are  made  up  approximately  as  follows : 

Albumins    68.5 

Albumose-like  bodies    21.6 

Nucleins    < 9-9 

A  characteristic  basic  body  known  as  spermine  is  present  in  small 
amount.  The  empirical  formula  C2H5N  has  been  given  to  it.  This 
substance  forms  a  combination  with  phosphoric  acid  which  sometimes 
separates  in  crystalline  form  on  evaporation  of  the  fluid.  The  charac- 
teristic odor  of  the  discharged  secretion  is  said  to  be  due  to  partial 
decomposition  of  the  base. 

The  spermatozoa  are  relatively  stable  bodies  and  resist  the  action  of 
chemical  reagents  to  a  remarkable  degree.  The  heads  of  spermatozoa 
consist  largely  of  nuclein  compounds  while  the  tails  contain  other  pro- 
teins, cholesterol,  fat  and  lecithin.  The  ash  content  of  the  whole  is 
relatively  high  and  is  rich  in  potassium  phosphate. 

BRAIN  AND  NERVE  SUBSTANCES.     CEREBRO-SPINAL 

LIQUID. 

These  tissues  contain  several  peculiar  compounds  of  which  our 
knowledge  is  limited,  largely  because  of  the  great  difficulty  in  separa- 
tion. The  solid  matter  of  the  brain  contains  globulins,  nucleo-proteids, 
cholesterol,  lecithin,  fatty  bodies  and  complex  compounds  not  found 
elsewhere.  Various  soluble  extractives,  somewhat  similar  to  those 
from  muscular  tissue,  are  also  present. 

Protagon.  This  has  been  assumed  to  be  an  important  constituent 
of  the  white  substance  of  the  brain,  which  has  this  elementary  compo- 
sition, according  to  Gamgee:  C  66.4,  H  1.07,  N  2.4,  P  1.07.  But  as 
others  writers  report  rather  widely  different  figures  it  is  likely  that  the 
pure  substance  has  not  yet  been  isolated.  As  extracted  by  means  of 
85  per  cent  alcohol  at  45  °  from  the  minced  brain,  and  purified  by 
crystallization  and  washing  with  ether,  it  is  obtained  as  a  white  powder 


MUSCLE    AND    ITS    EXTRACTIVES.  277 

practically  insoluble  in  cold  ether  or  alcohol  and  not  properly  soluble  in 
water.  With  much  water  it  finally  yields  a  gelatinous  liquid,  which 
suffers  decomposition  readily. 

Notwithstanding  the  bulky  literature  which  has  accumulated  in  the 
discussion  of  this  substance,  its  exact  nature  is  not  yet  known.  All 
recent  investigations  seem  to  show  that  it  is  a  mixture  of  a  number  of 
bodies.  By  treatment  with  certain  solvents,  or  by  gentle  cleavage,  it 
is  possible  to  separate  a  group  of  phosphatides,  similar  to  some  of  the 
lecithin  bodies,  and  a  group  of  substances  free  from  phosphorus,  but 
containing  nitrogen.  Cerebrin  and  cerebron  are  names  given  to  two 
of  these  products.     Of  the  functions  of  these  little  is  known. 

In  the  white  substance  of  the  spinal  marrow  the  so-called  protagon 
is  abundant.  In  degeneration  changes  in  the  tissues  of  the  nervous 
system  it  is  probably  this  compound  which  suffers  the  greatest  altera- 
tion, with  the  production  of  neurine  with  marked  toxic  properties.  It 
is  likely  that  the  neurine  comes  from  a  lecithin  body  as  one  of  the 
groups  in  the  protagon  complex,  and  that  these  reactions  will  prove  of 
great  importance  in  pathological  study. 

It  is  also  known  that  complex  sulphur  compounds  are  present  in  the 
brain  tissue,  but  little  is  known  of  their  reactions. 

Cerebrospinal  Liquid.  This  is  a  thin,  watery  liquid  of  which  only 
a  few  partial  analyses  have  been  recorded.  Its  general  character  is 
shown  by  these  figures  recently  given  by  Zdorek : 

1,000  parts  by  weight  contain 

Dry  substance    10.45 

Organic    2.09 

Inorganic    8.36 

Proteins    0.77 

Chlorine     4.24 

Sodium    oxide    4.29 

The  organic  substance  includes  traces  of  fats,  lecithin,  cholesterol 
and,  pathologically,  choline  or  neurine.  Common  salt,  however,  is  the 
main  solid  substance  in  solution. 

MUSCLE   AND    ITS   EXTRACTIVES. 

A  large  part  of  the  solid  portion  of  the  body  is  made  up  of  muscular 
tissue.  A  knowledge  of  the  composition  of  this  tissue  is  of  the  highest 
importance,  especially  since  some  of  the  fundamental  chemical  reactions 
of  the  animal  organism  take  place  within  the  cells  of  the  muscles.  For- 
tunately we  have  fairly  satisfactory  information  on  some  of  the  points 


278  PHYSIOLOGICAL    CHEMISTRY. 

of  interest  here,  as  numerous  analyses  have  been  made  of  the  muscles 
and  of  the  liquid  which  may  be  extracted  in  various  ways  from  them. 
The  dry  part  of  the  muscle  is  made  up  largely  of  proteins  of  which 
several  are  present ;  in  the  muscle  plasma  there  are  at  least  five  accord- 
ing to  Halliburton.  In  addition  to  these  bodies  there  are  a  number  of 
so-called  extractives  which  play  an  important  part. 

GENERAL  COMPOSITION  OF  MUSCLE. 

The  following  figures  represent  approximately  the  average  compo- 
sition of  the  fresh  muscle  dissected  free  from  visible  fat. 

Water    76  per  cent. 

Solids     24 

Proteins  (true)    17-6 

Collagen  substance  3-o 

Fat,   interstitial    1.5 

Flesh    bases    0.2 

N-f ree  extractives    0.4 

Salts    1.3 

The  Muscle  Proteins.  It  is  not  possible  to  give  a  perfectly  clear 
account  of  all  these  bodies  at  the  present  time,  as  the  products  obtained 
by  different  investigators  vary  with  the  details  of  the  extraction 
methods  employed.  The  more  important  constituents  commonly  rec- 
ognized are  indicated  in  the  following  paragraphs.  By  washing  out 
the  blood  from  living  muscle  by  physiological  salt  solution  (transfu- 
sion), dissecting  it,  grinding  it  to  a  pulp  and  pressing  very  strongly  a 
clear  yellowish  liquid  is  obtained  which  is  called  muscle  plasma.  The 
ordinary  dead  muscle  treated  in  the  same  manner  yields  a  different 
liquid  which  may  be  called  muscle  serum.  The  plasma  has  an  alka- 
line reaction  and  is  distinguished  by  the  property  of  spontaneous 
coagulation. 

The  term  myosin  was  formerly  applied  to  the  solidified  or  coagulated 
body  as  a  whole,  but  experiment  shows  that  two  things  at  least  are 
here  present.  One  of  these  is  called  musculin,  or  by  some  authors, 
myosin  proper,  while  the  other  product  is  known  as  myogen.  The 
musculin,  or  myosin,  coagulates  at  about  470,  while  for  myogen  the 
coagulating  temperature  is  about  560. 

The  two  substances,  musculin  and  myogen,  differ  also  in  their  pre- 
cipitation properties.  The  first  is  precipitated  from  solution  by  adding 
ammonium  sulphate  to  make  up  28  per  cent;  from  the  filtrate  the 
myogen  may  be  thrown  down  by  adding  the  sulphate  to  saturation, 
and  is  found  to  make  up  about  80  per  cent  of  the  plasma  protein. 


MUSCLE    AND    ITS    EXTRACTIVES.  279 

The  serum  left  after  the  formation  of  the  plasma  coagulum  usually 
contains  a  little  soluble  albumin.  This  may  be  normal  to  the  muscle 
substance,  or  it  may  be  due  to  the  blood  not  perfectly  removed  by  the 
preliminary  washing.  At  any  rate  the  plasma  consists  essentially  of 
the  two  myosin  bodies. 

After  separation  of  the  plasma  what  may  be  called  the  stroma  re- 
mains. This  is  mainly  albuminous,  but  its  exact  nature  is  not  known. 
The  sarcolemma  portion  of  the  muscle  fiber,  which  by  weight  makes 
up  but  a  small  part  of  the  whole,  appears  to  belong  to  the  albumoid 
group  of  proteins,  resembling  elastin.  It  has  been  shown  in  an  earlier 
chapter  that  from  ordinary  dead  muscle,  as  represented  by  lean  meat, 
a  considerable  amount  of  "  myosin  "  may  be  separated  by  extracting 
with  a  weak  solution  of  ammonium  chloride.  What  remains  does  not 
agree  fully  with  the  stroma  left  on  pressing  out  the  plasma  of  the 
fresh  muscle,  but  contains  approximately  the  same  substances.  By 
this  method  of  separation  the  insoluble  stroma  portion  is  much  larger 
than  the  soluble  or  "  myosin  "  portion.  The  latter  may  amount  to  7 
or  8  per  cent  of  the  weight  of  the  muscle  in  the  mean. 

Collagen.  As  given  in  the  above  table  this  refers  to  the  binding 
substance  holding  the  muscle  fibers  together  and  includes  the  sarco- 
lemma. It  is  insoluble  in  cold  water,  but  swells  and  disintegrates 
finally  in  boiling  water. 

Fat.  After  removing  all  visible  fat  from  the  dissected  muscle, 
analyses  still  show  a  small  amount  remaining.  This  must  therefore 
be  associated  with  the  minute  structure  of  the  fibrils. 

Flesh  Bases.  A  number  of  very  remarkable  substances  are 
included  here.  They  are  sometimes  described  as  the  nitrogenous 
extractives.  The  most  abundant  of  these  bodies  is  creatine  or  methyl- 
guanidine  acetic  acid ;  some  of  the  purine  bases  are  also  present.  A 
brief  description  of  these  substances  may  be  given. 

Creatine,  C4H9N302,  may  be  represented  structurally  by  the 
formula 

H-N=C<    CH, 

wCH,COOH 

It  is  found  in  all  muscles  and  is  probably  a  product  of  metabolism,  but 
the  method  of  its  formation  is  not  yet  known.  Being  readily  soluble 
in  warm  water,  and  in  about  75  parts  of  water  at  the  ordinary  tem- 
perature its  extraction  from  muscle  is  easy.  When  the  solution  is 
boiled  with  dilute  hydrochloric  acid,  through  a  long  period,  a  molecule 
of  water  is  split  off  and  the  anhydride  creatinine  is  left.     This  is  a 


280  PHYSIOLOGICAL    CHEMISTRY. 

normal  urinary  constituent  and  will  be  described  later.  When  boiled 
with  alkali  solution,  especially  baryta  water,  creatinine  undergoes  a 
complete  cleavage  into  urea  and  sarcosine,  which  relation  is  an  inter- 
esting one  and  has  suggested  a  possible  derivation  of  the  urinary  urea. 
Creatinine  may  be  readily  crystallized  from  water  solution.  It  was 
formerly  made  for  experiment  directly  from  meat.  It  is  best  secured 
from  certain  crystalline  residues  occurring  as  by-products  in  the  manu- 
facture of  "  beef  extract,"  referred  to  below. 

Carnine.  The  amount  of  this  in  muscle  is  very  small,  but  it  may 
be  recognized  in  beef  extract.  It  bears  some  relation  in  structure  to 
hypoxanthine,  and  has  been  given  the  formula  C7H8N4Os. 

Comparatively  recently  several  other  crystalline  products  have  been 
isolated  from  meat  extracts.  Among  these  carnosine  and  carnitine 
are  perhaps  the  most  important. 

The  Xanthine  Bodies.  These  constitute  a  peculiar  group  of 
great  importance  because  of  their  relation  to  uric  acid  and  other  products 
of  metabolism.  Traces  of  several  of  them  have  been  recognized  in  the 
muscular  juices ;  in  a  later  chapter  the  structure  and  properties  of  the 
substances  will  be  discussed  in  connection  with  uric  acid.  Traces  of 
urea  are  also  found  in  the  muscles. 

The  Nitrogen-Free  Extractives.  The  muscular  juices  hold  dis- 
solved a  number  of  compounds  which  contain  no  nitrogen,  some  of 
which  are  very  important.  The  chief  of  these  are  glycogen,  inosite, 
glucose  and  lactic  acid. 

Glycogen.  The  chemical  relations  of  glycogen  have  been  dis- 
cussed already  in  earlier  chapters.  The  glycogen  as  found  in  the 
muscles  comes  from  the  liver,  being  transported  there  by  the  blood,  and 
in  part  is  probably  formed  in  the  muscles  by  the  same  kind  of  an 
enzymic  action  which  leads  to  its  synthesis  in  the  liver.  The  liver  is 
capable  of  storing  up  a  large  weight  of  the  reserve  substance  in  a  small 
space.  The  amount  stored  in  an  equal  weight  of  muscle  is  small,  but 
taking  the  muscles  of  the  body  as  a  whole  the  glycogen  content  is  con- 
siderable, reaching  a  hundred  grams  or  more. 

It  is  probably  through  this  glycogen  that  the  muscle  is  capable  of 
doing  its  work.  Through  enzymic  hydration  the  glycogen  becomes 
sugar,  possibly  maltose  and  then  glucose,  and  the  potential  energy  of 
this  is  liberated  by  oxidation  to  water  and  carbon  dioxide  ultimately. 
The  oxidation  may  not  be  direct;  in  all  probability  there  are  several 
transformation  products  before  the  final  stages  are  reached.  But  the 
energy  transformation  is  the  same  whatever  the  intermediate  steps  may 
be.     The  importance  of  the  glycogen  and  related  bodies  in  this  direc- 


MUSCLE    AND    ITS    EXTRACTIVES.  251 

tion  will  be  pointed  out  in  a  following  chapter.  It  may  be  recalled  that 
in  these  oxidation  processes,  where  sugar  is  concerned,  a  muscle  enzyme 
and  a  pancreas  enzyme  seem  to  be  both  necessary. 

While  glycogen  in  the  muscles  must  come  mostly  from  sugars,  either 
directly  or  through  the  liver,  there  is  also  some  evidence  that  it  may 
come  in  part  from  other  substances,  especially  from  proteins.  Animal 
experiments  have  shown  apparently  a  storing  of  glycogen  from  a  pro- 
tein diet  after  previous  starvation  had  exhausted  the  reserve  in  store. 
In  the  breaking  down  of  some  proteins  it  has  been  shown  that  certain 
carbohydrate  groups  are  liberated ;  it  is  doubtless  these  which  undergo 
synthesis  to  form  at  least  part  of  the  glycogen,  and  from  this  stand- 
point the  behavior  of  protein  as  a  glycogen  factor  is  not  so  hard  to 
understand. 

The  glycogen  content  of  the  muscles  of  different  animals  is  var- 
iable; in  the  flesh  of  the  horse  it  is  relatively  high,  amounting  often 
to  over  i  per  cent.  As  the  muscle  glycogen  is  not  altered  rapidly  in 
the  dead  organ,  as  is  the  liver  glycogen,  the  presence  of  the  substance 
in  horse-flesh  sausage  may  be  quite  readily  recognized.  Methods  have 
been  devised  for  the  identification  of  horse-flesh,  sold  for  food,  based 
on  these  facts.  Glycogen  may  be  extracted  from  the  muscles  by  the 
general  method  given  for  the  liver  in  an  earlier  chapter;  the  chemical 
and  optical  properties  may  be  used  for  the  final  identification. 

Inosite.  This  substance  has  the  empirical  formula  C6H1206  -f-  H20 
and  was  long  spoken  of  as  muscle  sugar.  It  is  not  a  true  carbohydrate, 
however,  but  an  aromatic  product  C6H6(OH)G,  that  is,  hexahydroxy- 
benzene.  The  amount  found  in  muscle  is  very  small  and  how  it  is 
derived  is  not  known ;  but  it  is  not  peculiar  to  these  tissues,  as  it  occurs 
in  other  organs  of  the  body  and  also  in  many  vegetable  substances.  It 
may  be  extracted  from  muscles  without  much  trouble  and  when  pure 
is  found  to  be  a  white  crystalline  powder  melting  at  about  2200.  It 
is  very  soluble  in  water,  to  which  a  sweetish  taste  is  given,  and  in  pres- 
ence of  alkali  is  not  a  reducing  agent  for  metallic  solutions.  Although 
the  usual  structural  formula  does  not  show  an  asymmetric  carbon  atom 
the  substance  is  optically  active  and  exhibits  a  strong  rotation,  both 
right  and  left  forms  being  known. 

Glucose.  From  what  was  said  above  about  the  transformation  of 
glycogen  it  is  not  surprising  that  a  small  amount  of  sugar  should  be 
found  in  the  muscles ;  both  maltose  and  glucose  have  been  detected. 

Lactic  Acid.  Several  forms  of  this  acid  are  known,  but  that  occur- 
ring in  the  muscle  is  the  dextrorotatory  paralactic  or  sarcolactic  acid, 
O.I  [«08.      It  is  one  of  the  a-hydroxypropionic  acids.     There  has  been 


282  PHYSIOLOGICAL    CHEMISTRY. 

much  speculation  as  to  the  source  of  this  acid  in  the  body,  but  it  seems 
most  rational  to  regard  it  as  derived  from  the  glycogen  or  sugar  by  a 
comparatively  simple  cleavage.  It  is  also  possible  that  in  the  katabolic 
reactions  of  proteins  lactic  acid  may  result  from  a  splitting  of  the 
carbohydrate  group.  The  acid  is  not  very  readily  detected  in  the  living 
muscle  because  it  is  probably  oxidized  or  removed  too  rapidly  by  the 
fluid  circulation.  In  the  dead  muscle,  however,  it  may  accumulate  to 
the  extent  of  half  a  per  cent  or  more.  The  living  muscle  shows  a 
neutral  or  slightly  alkaline  reaction,  while  in  the  dead  muscle  the 
increase  of  lactic  acid  changes  the  reaction. 

The  lactic  acid  of  the  muscle  probably  results  from  an  enzymic 
cleavage.  In  the  aseptic  autolysis  of  liver  paralactic  acid  has  been 
recognized  among  the  products,  and  this  fact  shows,  at  least,  the  possi- 
bility of  such  a  formation.  The  amount  of  lactic  acid  formed  in  the 
muscle  seems  to  be  greatest  during  working  periods,  which  is  true  also 
of  the  final  products  of  katabolism.  The  acid  may  simply  represent 
a  stage  in  the  gradual  breaking  down,  whether  we  consider  a  carbohy- 
drate or  protein  as  the  parent  substance.  We  should  expect  therefore 
an  increase  in  the  muscle  acid  if  the  oxidation  processes  of  the  body 
are  hindered  or  retarded,  while  at  the  same  time  protein  or  sugar 
decomposition  is  increased,  or,  at  any  rate,  not  diminished.  In  the 
dead  muscle  the  enzymic  formation  of  lactic  acid  doubtless  continues 
long  after  the  oxidation  reaction  ceases,  and  this  is  probably  the  main 
reason  for  the  ready  detection  in  the  muscle  after  death. 

The  pure  acid  occurs  as  a  thickish  liquid  miscible  with  water.  It 
forms  salts  which  are  mostly  readily  soluble.  The  zinc  and  calcium 
salts  crystallize  well  and  are  hence  prepared  for  identification.  The 
pure  liquid  shows  a  right  hand  optical  rotation,  with  [a]D  =  about  30. 
The  result  is  not  constant  because  of  the  difficulty  of  preparing  con- 
centrated solutions  free  from  anhydride  or  lactide.  The  rotation  of 
the  salts,  on  the  contrary,  is  to  the  left. 

The  Inorganic  Salts.  Although  making  up  not  much  over  1  per 
cent  of  the  weight  of  the  moist  muscle,  these  salts  are  extremely  im- 
portant. Of  dry  substance  the  salts  constitute  5  per  cent  or  more. 
The  salts  are  usually  estimated  from  the  ash  left  in  burning  the  mus- 
cle; this  gives  of  course  no  correct  idea  of  how  they  are  combined  in 
the  living  muscle,  but  is  the  only  method  available.  In  the  living 
muscle  many  of  the  inorganic  elements  are  doubtless  in  chemical  union 
with  proteins  or  other  organic  groups,  while  in  the  derived  ash  we 
have  chlorides,  phosphates,  sulphates  or  carbonates.  A  carbonate  is 
probably  formed  during  the  combustion  of  organic  acids  and  corre- 


MUSCLE    AND    ITS    EXTRACTIVES.  283 

sponds  to  no  simple  preexisting  compound.  Phosphorus  and  sulphur 
of  proteins  furnish  phosphates  and  sulphates.  The  analyses  of  ash 
made  disclose  very  different  results,  but  mean  values  may  be  given  to 
show  the  general  approximate  composition.  In  the  calculation  car- 
bonic acid  is  not  considered.  The  table  below  is  from  the  Konig 
collection. 

K.0   37-04 

Na,0    10.14 

CaO   2.42 

MgO    3-23 

Fe203  0.44 

P,05    4120 

S03    0.98 

CI   4-66 

Si02  0.69 

From  the  table  it  appears  that  potassium  phosphate  is  the  most 
abundant  substance  in  the  ash.  Much  of  this  doubtless  preexists  in 
the  muscle  juices,  while  a  small  portion  is  of  oxidation  origin.  The 
small  sulphate  content  is  probably  due  to  protein  sulphur  fully  oxidized 
in  the  combustion.  In  the  past  too  little  attention  has  been  given  to 
the  mineral  constituents  of  the  body,  it  being  commonly  assumed  that 
they  represent  "waste"  or  "ash"  only.  But  the  newer  applications 
of  chemistry,  especially  physical  chemistry,  to  physiology  have  dis- 
closed the  fact  that  the  inorganic  salts  are  especially  concerned  in  the 
proper  maintenance  of  many  of  the  body  functions.  The  balanced 
osmotic  pressure  of  the  body  fluids  is  largely  a  function  of  the  salt 
content,  and  variations  here  are  of  great  importance.  The  mineral 
salts  are  the  carriers  of  electric  charges  in  the  body  and  as  such  seem 
to  have  important  duties  to  perform. 

EXTRACT  OF  MEAT. 

By  boiling  lean  meat  with  water  the  soluble  constituents  are  dis- 
solved, producing  an  extract.  When  this  is  concentrated  to  a  paste 
the  article  known  commercially  as  "  Extract  of  Meat "  results.  The 
article  was  first  made  in  quantity  in  South  America  to  utilize  the  car- 
casses of  cattle  slaughtered  for  the  hides,  but  later  the  manufacture 
was  introduced  elsewhere,  and  generally  to  utilize  certain  waste  or 
by-products  in  the  meat  industries.  At  first  the  extract  was  assumed 
to  possess  food  value  in  a  high  degree,  but  after  a  time,  as  the  chem- 
istry of  the  proteins  and  their  derivatives  became  better  understood, 
this  notion  was  gradually  abandoned.  Lean  meat,  muscle,  is  employed 
practically  in  the  process ;  hence  little  or  no  fat  can  be  present.     At  the 


284  PHYSIOLOGICAL    CHEMISTRY. 

boiling  temperature  nearly  the  whole  of  the  proteins  are  coagulated 
and  are  filtered  out.  A  little  gelatin  remains,  but  the  food  value  of 
this  is  of  minor  importance.  Unless  the  boiling  is  greatly  prolonged 
the  extract  must  therefore  contain  essentially  the  meat  bases  and  other 
extractives  referred  to  above,  and  the  actual  nutritive  value  of  these  is 
low,  in  the  case  of  the  bases  being  nil.  On  prolonged  boiling,  how- 
ever, a  small  portion  of  the  original  protein  seems  to  pass  over  into 
the  soluble  form  of  albumose,  which  is  therefore  found  in  some  ex- 
tracts. Finally,  the  phosphates  and  other  inorganic  salts,  being  largely 
soluble,  pass  into  the  extract  and  constitute  a  considerable  part  of  the 
finished  pasty  product. 

In  this  country  "  extract "  is  made  by  concentrating  the  broth  resulting  from  the 
boiling  of  beef  as  a  step  in  the  canning  process.  Large  quantities  of  meat  being 
boiled  in  the  same  water,  it  becomes  rich  in  the  "  extractives  "  and  is  finally  boiled 
down  to  the  usual  pasty  condition.  Before  the  concentration  is  complete  the  liquid 
is  filtered  and  skimmed  and  therefore  leaves  a  residue  free  from  fat  or  fiber 
Roughly  speaking  the  paste  extract  has  about  this  composition: 

Water 20 

Salts    20 

Organic  substances    60 

Numerous  analyses  have  been  made  of  some  of  the  commercial  extracts,  but  the 
methods  employed  have  not  always  been  delicate  enough  to  furnish  trustworthy 
information.  This  is  especially  true  as  regards  the  amounts  of  so-called  peptone 
and  albumose  present,  for  which  the  definitions  have  not  been  fairly  uniform  until 
comparatively  recently.  The  recognized  relations  of  these  substances  are  explained 
in  the  chapter  on  protein  compounds.  Analyses  made  by  the  older  methods  were 
generally  reported  as  showing  more  or  less  "  peptone "  when,  according  to  the 
present  views,  "  albumose  "  is  meant.  The  following  figures  may  be  taken  as  repre- 
senting approximately  the  average  composition  of  typical  samples  of  American  meat 
extract : 

Water    20.0 

Inorganic  salts   (ash)    22.5 

Albumose  (and  gelatin)    16.5 

Flesh   bases,    etc 26.4 

N-f ree  extractives 14.6 

According  to  these  results  the  food  value  of  the  extract  would  be  measured  by 
the  nitrogen-free  extractives  and  the  albumose  and  gelatin  fractions.  In  some  kinds 
of  extract  the  flesh  bases  and  related  bodies  are  much  higher  than  here  given,  with 
corresponding  diminution  in  the  other  organic  constituents.  The  real  value  of  these 
extracts  lies  mainly  in  other  directions,  however.  They  contain  the  flavoring  and 
stimulating  portions  of  the  meat,  and  should  not  be  considered  so  much  as  foods 
as  additions  to  foods.  Added  to  vegetables  they  impart  an  agreeable  taste  and 
doubtless  serve  a  very  useful  purpose  in  stimulating  appetite  for  substances  not  in 
themselves  possessing  much  flavor.  In  their  action  the  basic  and  similar  substances 
in  the  meat  extracts  may  be  perhaps  fairly  compared  with  the  alkaloids  in  tea  and 
coffee,  which,  experience  shows,  have  a  real  value.     Large  amounts  of  the  extracts 


BONE    AND    GELATIN.  285 

cannot  be  used,  however,  as  foods,  because  of  the  presence  of  the  large  percentages 
of  alkali  phosphates  and  other  salts.  A  few  simple  experiments  may  be  made  to 
show  some  of  the  properties  of  the  common  commercial  extracts. 

Experiment.  Heat  a  little  of  the  solid  extract  on  a  piece  of  porcelain  until  it 
is  reduced  to  a  char.  Extract  this  with  dilute  nitric  acid,  filter  and  divide  the 
filtrate  into  two  portions.  In  one  test  for  phosphates  by  the  addition  of  ammonium 
molybdate  and  in  the  other  for  potassium  salts  by  the  flame  test.  Both  reactions 
should   be   very   distinct. 

Experiment.  Dissolve  20  grams  of  extract  in  water  to  make  about  200  cubic 
centimeters.  A  nearly  clear  solution  should  be  obtained,  showing  absence  of  fat  or 
coagulated  protein.  To  a  few  cubic  centimeters  add  enough  weak  acetic  acid  to  give 
a  slight  reaction,  and  boil.  If  a  precipitate  forms,  which  is  rarely  the  case,  albumin 
is  shown. 

With  50  cc.  of  the  liquid  make  the  albumose  test.  Add  to  it  finely  powdered  zinc 
sulphate  as  long  as  it  dissolves  on  stirring.  On  saturating  the  solution  completely 
a  flocculent  precipitate  gradually  settles.  This  is  essentially  the  "  albumose "  frac- 
tion and  may  contain  a  little  gelatin.  After  24  hours  filter,  and  test  the  filtrate  for 
peptone  by  the  biuret  reaction ;  this  is  generally  negative. 

Use  the  remainder  of  the  original  solution  for  the  recognition  of  creatine.  Add 
to  it  carefully  a  solution  of  basic  acetate  of  lead  as  long  as  a  precipitate  forms. 
This  will  carry  down  phosphates,  sulphates  and  other  compounds  forming  insoluble 
combinations  with  it,  but  not  creatine.  A  slight  excess  of  the  lead  must  be  added 
to  insure  complete  precipitation.  This  can  be  determined  by  allowing  the  first 
formed  precipitate  to  settle  and  adding  more  reagent  as  necessary.  Finally  filter, 
and  remove  the  excess  of  lead  by  passing  in  hydrogen  sulphide.  Filter  again,  and 
remove  as  much  as  possible  of  the  excess  of  sulphide  used,  by  shaking.  Then  con- 
centrate the  liquid  to  a  small  volume  by  slow  evaporation  on  the  water-bath  and 
allow  it  to  stand  a  day  or  more  in  a  cool  place  for  crystallization  of  the  creatine. 
Pour  off  the  supernatant  liquid  and  wash  the  fine  crystals  obtained  with  a  little 
strong  alcohol  in  which  creatine  is  but  slightly  soluble. 

Experiment.  Dissolve  the  creatine  in  a  little  hydrochloric  acid  and  evaporate 
the  solution  slowly  to  dryness  on  the  water-bath.  This  action  converts  creatine  into 
creatinine.  Dissolve  the  residue  in  a  little  water  and  divide  the  solution  into  two 
parts.  To  one  add  a  solution  of  zinc  chloride,  which  produces  a  white  crystalline 
precipitate  containing  the  creatinine-zinc  chloride,  (C4H7N30)2ZnCl2.  The  character 
of  the  crystals  can  be  seen  under  the  microscope.  To  the  other  part  of  the  solu- 
tion add  a  few  drops  of  a  dilute  solution  of  sodium  nitroprusside  and  then,  drop 
by  drop,  dilute  solution  of  sodium  hydroxide.  This  gives  a  ruby  red  color  which 
fades  to  yellow.  Add  enough  acetic  acid  to  change  the  reaction  and  warm.  The 
color  becomes  green  and  finally  blue.  This  is  known  as  Weyl's  reaction.  The  blue 
color  finally  obtained  is  Prussian  blue. 

A  further  very  delicate  reaction  for  creatinine  is  given  later,  in  the  chapter  on 
urine  analysis. 

Experiment.  The  mother  liquor  left  after  crystallizing  the  creatine  contains 
traces  of  xanthine  bases.  Add  enough  ammonia  to  give  an  alkaline  reaction  and 
filter.  Then  add  a  few  drops  of  ammoniacal  solution  of  silver  nitrate  which  pre- 
cipitates the  several  substances  in  flocculent  form. 

BONE   AND    GELATIN. 

In  the  moist  bone  as  it  exists  in  the  body  the  water  and  solids  are, 
in  the  mean,  in  about  the  proportion  of  one  to  two.     In  very  young 


286  PHYSIOLOGICAL    CHEMISTRY. 

persons,  however,  the  water  is  in  greater  excess,  while  with  age  the 
solids  increase.  The  solid  matter  consists  roughly  of  I  part  of  organic 
matter  to  2  of  mineral. 

THE  ORGANIC  MATTER  OR  OSSEIN. 

The  crude  organic  substance  in  the  bone  is  commonly  called  ossein ; 
it  may  be  extracted  with  hot  water  and  forms  a  gelatinous  mass  on 
cooling.  But  fuller  investigations  show  that  this  ossein  is  not  a  single 
substance,  as  several  different  constituents  may  be  separated  by  proper 
solvents.  These  are,  however,  closely  related  substances  and  for  our 
present  purpose  they  may  all  be  considered  as  practically  identical  with 
the  collagen  or  glue-forming  substance  of  the  connective  tissues.  The 
conversion  of  the  ossein  or  collagen  into  gelatin  appears  to  be  a  hydra- 
tion process,  as  at  a  higher  temperature  the  reverse  operation  takes 
place.  The  preparation  and  properties  of  bone  gelatin  may  be  illus- 
trated experimentally: 

Experiment.  Clean  a  long,  slender  bone  (best,  a  rib),  and  immerse  it  in  dilute 
hydrochloric  acid  of  about  ten  per  cent  strength.  Let  it  remain  several  days.  At 
the  end  of  this  time  remove  the  bone  from  the  acid  and  observe  that  it  has  lost 
its  rigidity  and  has  become  very  flexible.  It  may  be  even  possible  to  tie  it  in  a 
knot.  Wash  the  elastic  mass  several  times  in  fresh  water  to  remove  all  the 
hydrochloric  acid,  then  with  a  little  dilute  sodium  carbonate  solution  followed  by 
more  water,  and  finally  boil  it  with  a  small  amount  of  pure  water.  By  heating  it 
long  enough  the  ossein  becomes  converted  into  gelatin,  which  solidifies,  on  cooling, 
to  a  jelly. 

By  boiling  the  bone  ossein  under  pressure  the  formation  of  the  gelatin  is  very 
much  hastened. 

The  solution  as  obtained  above  may  be  used  for  tests  such  as  were  described  in 
Chapter  V,  under  Gelatin. 

THE  MINERAL  MATTER  IN  BONES. 

We  are  not  able  to  say  exactly  how  the  mineral  elements  are  com- 
bined in  the  moist  fresh  bone.  Our  knowledge  of  these  combinations 
is  practically  limited  to  what  we  can  learn  by  a  study  of  the  residue 
left  on  burning  the  bone  completely,  known  as  boneash.  This  is  a 
white  powder  containing  the  non-volatile  compounds,  of  which  calcium 
phosphate  is  the  most  important.  The  following  table  shows  the  aver- 
age composition  of  human  boneash : 

Calcium   phosphate    85.7  per  cent. 

Magnesium  phosphate  1.5         " 

Calcium    carbonate     11.0         " 

Calcium  fluoride  and  chloride 1.0         " 

Ferric    oxide    0.8         " 

100.0 


BONE    AND    GELATIN.       CARTILAGE.  287 

The  presence  of  calcium,  magnesium  and  phosphoric  acid  may  be 
shown  in  the  weak  hydrochloric  acid  extract  of  the  bone  described 
above. 

Experiment.  To  a  few  cubic  centimeters  of  the  filtered  solution  add  some 
ammonium  molybdate  solution.  In  a  short  time  a  yellow  precipitate  appears,  indi- 
cating presence  of  a  phosphate,  as  familiar  to  the  student  from  the  reactions  of 
qualitative  analysis. 

Experiment.  To  a  few  cubic  centimeters  of  the  solution  add  solution  of  sodium 
acetate  until  a  distinct  odor  of  acetic  acid  persists.  Then  add  some  solution  of 
ammonium  oxalate,  which  produces  a  white  precipitate  of  calcium  oxalate. 

Experiment.  To  another  portion  of  the  hydrochloric  acid  solution  add  am- 
monia until  a  good  alkaline  reaction  is  obtained.  A  white  precipitate  of  calcium 
and  magnesium  phosphates  settles  out.  Filter,  and  to  the  filtrate  add  some  am- 
monium oxalate  solution.  A  further  precipitate  appears.  This  is  calcium  oxalate 
and  proves  that  the  original  solution  contains  calcium  in  excess  of  that  combined 
as  phosphate.    The  calcium  of  the  carbonate,  fluoride  and  chloride  appears  here. 

Experiment.  To  detect  the  small  amount  of  magnesium  requires  greater  care. 
To  another  and  relatively  large  portion  of  the  acid  solution  add  enough  ammonia 
to  give  an  alkaline  reaction,  and  then  acidify  slightly  with  acetic  acid.  This  dis- 
solves everything  except  ferric  phosphate,  which  may  be  filtered  off  and  tested  for 
iron.  To  the  filtrate  add  enough  ammonium  oxalate  to  precipitate  all  the  calcium 
as  oxalate.  Separate  this  after  long  standing  by  means  of  close-grained  filter 
paper.  In  the  clear  filtrate  the  magnesium  may  be  thrown  down  with  the  phos- 
phoric acid  still  present,  by  the  addition  of  ammonia  water  in  slight  excess. 

Bone  Marrow.  The  pure  marrow  consists  largely  of  fat  in  which 
olein  is  abundant;  cholesterol  is  present  and  some  nitrogenous  extrac- 
tive substances,  which,  however,  have  not  been  very  thoroughly 
examined. 

CARTILAGE. 

Collagen  is  probably  the  most  abundant  substance  in  the  cartilagi- 
nous tissue  where  it  exists  mixed  or  combined  with  several  other 
bodies,  of  which  these  have  been  described :  chondronmcoid,  chon- 
droitin-sulphuric  acid  and  an  albuminoid.  The  nature  of  crude  col- 
lagen has  been  explained,  and  in  Chapter  V  the  somewhat  obscure 
chemistry  of  the  chondroitin-sulphuric  acid  has  been  outlined.  Of  the 
nature  of  the  chondromucoid  little  is  known  definitely;  it  has  been 
held  by  some  writers  to  be  merely  a  combination  of  part  of  the  collagen 
with  the  salts  of  the  complex  ethereal  sulphuric  acid  mentioned,  while 
Morner,  who  first  described  it,  held  it  for  a  distinct  body  somewhat 
allied  to  mucin.  His  analyses  showed  C  47-3°>  H  6.42,  N  12.58, 
S  2.42,  O  31.28.  The  sulphur  is  probably  all  in  the  ethereal  combi- 
nation and  on  incineration  of  the  cartilage  the  ash  is  found  to  contain 
a  very  large  amount  of  alkali  sulphate. 

Chondromucoid  as  separated  is  insoluble  in  water  alone,  but  with  a 
little  alkali    forms  a  thick   solution,   which   is  precipitated  by  acids. 


288 


PHYSIOLOGICAL    CHEMISTRY. 


Stronger  acids  bring  about  a  cleavage  with  separation  of  the  chon- 
droitin-sulphuric  acid.  The  weak  alkali  solutions  are  precipitated  by 
metallic  salts,  but  most  of  the  other  protein  reactions  fail.  The 
ethereal  sulphate  group  seems  to  prevent  the  ordinary  precipitations. 
The  albuminoid  substance  is  not  well  characterized  but  is  insoluble 
in  water,  and  in  weak  acids  or  alkalies.  It  undergoes  gastric  diges- 
tion. This  protein  is  said  to  be  found  in  old  cartilage  only,  and  is 
absent  in  young  cartilage. 

KERATIN   BODIES. 

Compounds  of  the  keratin  group  occur  in  hair,  the  finger  'nails  and 
horn.  They  resemble  the  proteins  but  contain  rather  large  amounts 
of  sulphur,  as  shown  by  these  analyses,  which  are  of  keratin  from 
several  sources : 


Hair. 

Nails. 

Horn. 

c 

50.65 

6.36 

17.14 

20.85 

5.OO 

51.OO 

6.94 

17-51 

21-75 

2.8o 

6.80 

H 

N 

16.24 

22.51 

3-42 

0 

S  

The  sulphur  in  hair  is  in  part  loosely  combined  and  may  be  split  off 
easily  by  reagents,  alkalies  for  example.  The  ash  of  hair  is  rich  in 
sulphates  and  contains  also  silica  and  other  mineral  substances.  Much 
of  the  ash  may  be  removed  by  washing  the  hair  with  weak  acids,  fol- 
lowing treatment  with  ether  and  alcohol  to  remove  fatty  and  other 
soluble  substances.  The  purified  "  keratin  "  thus  secured  gives  results 
like  the  above  on  analysis. 

Horn  and  nails  contain  along  with  the  insoluble  keratin  insoluble 
salts,  mainly  phosphate  of  calcium,  which  stiffen  them.  From  very 
fine  horn  shavings  these  salts  may  be  dissolved  out  by  acids,  leaving  a 
soft  flexible  keratin. 


SECTION    IV. 

THE  END  PRODUCTS  OF  METABOLISM.     EXCRE- 
TIONS.    ENERGY  BALANCE. 

CHAPTER   XIX. 

THE  EXCRETION  OF  NITROGEN,   SULPHUR   AND   PHOSPHORUS. 

THE  URINE. 

Having  considered  in  the  foregoing  pages  the  substances  used  in  the 
nutrition  of  the  body,  the  agencies  of  nutrition,  and  the  general  char- 
acter of  the  products  formed,  we  come  now  to  a  short  study  of  the 
waste  products  rejected  by  the  body  after  it  has  assimilated  and  used 
the  nutrients  furnished  to  it.  The  food-stuff's  which  the  animal  can 
utilize  are  comparatively  complex,  but  consist  essentially  of  the  mem- 
bers of  the  three  groups,  the  fats,  carbohydrates  and  proteins.  The 
theoretically  simplest  waste  or  oxidation  products  of  these  are  nitro- 
gen, carbon  dioxide  and  water,  but  in  the  animal  organism  the  breaking 
down  does  not  go  so  far.  While  from  fats  and  carbohydrates  essen- 
tially only  water  and  carbon  dioxide  are  formed,  the  protein  metabo- 
lism is  not  carried  to  the  elimination  of  nitrogen,  but  ends  with  the 
formation,  largely,  of  urea,  a  body  in  a  way  related  to  the  theoretical 
end  products,  but  which  would  call  for  three  more  atoms  of  oxygen 
to  complete  oxidation. 

The  nitrogen  metabolism  involves  some  extremely  interesting  prob- 
lems which  are  still  far  from  complete  solution.  From  the  older  point 
of  view  urea  was  considered  the  one  normal  end  point  in  the  chain  of 
katabolic  reactions,  and  the  other  nitrogenous  bodies  found  in  the 
urine,  such  as  uric  acid  and  creatinine,  were  looked  upon  as  substances 
which  in  some  way  had  accidentally  escaped  the  fate  due  them.  This 
view  is  doubtless  incorrect,  as  we  have  good  reason  to  believe  that  uric 
acid  is  not  a  step  in  the  ordinary  protein  metabolism,  but  is  a  derivative 
of  certain  substances  only,  which  break  down  to  a  limited  degree. 
The  amount  of  uric  acid  which  could  be  formed  in  this  way  would  not 
be  very  large  at  most.  In  the  metabolism  of  nitrogen,  therefore,  a 
number  of  normal  end  products  must  be  considered  and  these  will  be 
discussed  in  the  next  few  pages. 

The  question  of  the  fundamental  changes  in  protein  before  the 
20  289 


29O  PHYSIOLOGICAL    CHEMISTRY. 

recognizable  end  products  are  reached  is  one  in  which  there  has  been 
a  great  deal  of  discussion.  In  a  general  way  Pflueger  assumed  that  all 
protein  actually  katabolized  must  first  be  built  up  into  a  part  of  the 
living  tissues,  from  the  absorbed  products  of  protein  digestion.  The 
cells  of  this  living  tissue  must,  therefore,  undergo  constant  and  far- 
reaching  changes,  since  the  body  is  able  to  dispose  of  some  hundreds 
of  grams  daily  of  protein  in  forced  feeding.  The  somewhat  older 
theory  of  Voit  assumes  that  the  absorbed  protein,  in  the  form  of  com- 
plex molecules,  from  the  intestinal  tract,  is  carried  along  by  the  blood 
in  dissolved  or  suspended  condition  to  certain  cells  or  tissues,  and  is 
then  broken  down  through  the  influence  of  forces  residing  in,  or  ema- 
nating from,  these  tissues.  This  protein  is  described  as  circulating 
protein,  and  before  destruction  does  not  become  an  integral  part  of  the 
actual  tissues  of  the  body.  Both  of  these  theories,  following  the  older 
views  of  the  conditions  under  which  protein  is  absorbed  after  diges- 
tion, assume  that  only  the  highly  complex  protein  structures  are 
capable  of  beginning  the  katabolic  change.  But  in  late  years  the  facts 
brought  out  by  the  investigations  of  Cohnheim,  Abderhalden  and 
others,  on  the  fate  of  protein  in  the  digestive  operations,  have  sug- 
gested very  different  views  regarding  the  general  course  of  this  nitrog- 
enous metabolism.  It  appears  probable  that  the  greater  part  of  the 
protein  of  the  food,  broken  down,  as  it  largely  is,  into  the  component 
amino  acid  complexes,  and  absorbed  as  such,  may  not  be  built  up  again 
into  structures  like  the  original,  but  may  be  at  once  hydrolyzed  and 
oxidized.  A  nitrogenous  fraction  may  be  separated  in  the  form  of 
ammonia  by  a  hydrolytic  cleavage,  to  be  further  converted  into  urea, 
while  the  residue,  rich  in  carbon  and  hydrogen,  would  suffer  ultimate 
oxidation  like  a  fat  or  sugar. 

-  This  general  view,  which  has  found  expression  notably  by  Cohnheim 
and  Folin,  does  not  call  for  the  building  up  of  great  masses  of  tissue 
protein,  or  even  for  the  circulating  protein  of  Voit.  There  is  recon- 
struction of  protein  only  insofar  as  it  is  needed  for  the  repair  of  wasted 
or  worn  out  tissues,  and  of  the  extent  of  this  we  know  but  little.  It 
is  probable  that  the  protein  of  the  tissues,  in  its  final  katabolism  may 
yield  some  products  different  from  those  produced  in  the  hydrolysis 
of  the  simply  absorbed  complexes.  A  study  of  the  urine  gives  us 
some  ideas  on  this  subject,  which  will  appear  in  what  follows. 

It  will  be  well  to  begin  with  the  consideration  of  the  urine  as  a  whole, 
as  all  these  substances  are  eliminated  through  that  channel. 


THE   URINE.  29I 

THE  GENERAL  COMPOSITION  OF  URINE. 

The  work  of  the  kidneys  in  the  discharge  of  the  urine,  or  more  prop- 
erly the  separation  of  its  constituents  from  the  blood,  is  usually  spoken 
of  as  one  of  excretion.     But  something  more  than  simple  elimination 
of  worthless  products  is  here  concerned ;  the  work  done  by  these  organs 
is  in  part  secretory,  as  certain  synthetic  reactions  are  beyond  question 
carried  out  here.     Years  ago  Bunge  and  Schmiedeberg  demonstrated 
the  synthesis  of  hippuric  acid  from  benzoic  acid  and  glycocoll  in  the 
kidney,   and  since  then  other  changes  have  been  brought  to  light. 
Further  than  this,  the  peculiar  mechanism  of  the  kidney  accomplishes 
another  very  remarkable  thing.     The  blood  circulating  through  the 
kidney  contains  valuable  material  to  be  saved  as  well  as  worthless  sub- 
stances to  be  rejected.     Toward  all  these  constituents  the  epithelial 
cells  of  the  kidney  tubules  exercise  a  sort  of  selective  treatment.     The 
proteins,  which  are  colloids,  are  retained  by  the  blood,  but  the  sugar, 
which  is  a  crystalloid,  and  very  soluble,  is  retained  also  unless  its  con- 
centration passes  a  certain  limit.     The  soluble  salts  are  in  part  passed 
through  the  kidneys  and  in  part  retained  by  the  blood,  with  the  final 
result  of  maintaining  a  very  nearly  constant  osmotic  pressure  in  that 
fluid.     How  this  is  done  we  cannot  say.     It  is  indeed  a  problem  of 
physiology  and  histology  rather  than  of  chemistry.     We  know  only 
this,  that  the  selective  absorption  and  control  of  the  blood  concentra- 
tion are  perfectly  automatic.     When  the  osmotic  pressure  of  certain 
constituents  is  increased  beyond  a  pretty  definite  limit,  the  filtering 
mechanism  in  the  kidney  for  those  constituents  becomes  active  and  the 
excess  is  allowed  to  pass.     The  simple  laws  of  diffusion  and  osmotic 
pressure  do  not  help  us  greatly  in  explaining  the  actions  of  the  kidneys 
where  the  flow  of  excreted  substances  is  usually  from  a  level  of  low 
concentration  to  one  of  higher.     Attempts  have  been  made  to  compare 
the  separating  medium  between  the  urine  and  the  blood  to  a  semi- 
permeable membrane,  but  the  comparison  is  very  imperfect  unless  the 
degree  of  impermeability  be  specially  limited  for  each  substance  passing 
from  the  blood  to  the  urine.     The  limitation  would  have  to  account 
for  a  concentration  of  salt  from  about  0.6  per  cent  in  the  blood  to  over 
1.0  per  cent  in  the  urine,  while  for  urea  the  concentration  would  change 
from  about  0.05  per  cent  or  lower  to  over  2.0  per  cent,  that  is,  forty 
fold.     Limitations  as  wide  as  these  render  the  comparison  of  little 
practical  service. 

Percentage  Variations.     It  is  not  possible  to  speak  of  the  mean 
strength  of  normal  urine  since  the  variations  are  extremely  irregular, 


292  PHYSIOLOGICAL    CHEMISTRY. 

depending  in  health  on  a  great  many  factors.  The  volume  excreted 
daily,  as  stated  in  the  books,  is  usually  given  much  too  high  for  the 
conditions  obtaining  in  the  United  States.  In  place  of  the  1,500  cc. 
as  found  in  most  of  the  foreign  works  we  should  take  1,150  to  1,200 
cc.  as  nearer  the  average  excretion  for  24  hours.  In  some  hundreds 
of  examinations  made  by  the  writer  in  the  last  few  years  on  people  of 
both  sexes  engaged  in  various  occupations  the  average  volume  comes 
within  these  limits. 

A  number  of  complete  analyses  of  urine  are  found  in  the  literature,  but  in  most 
of  them  the  uric  acid  content  is  placed  too  low  because  of  the  faulty  methods  of 
determination  formerly  employed.  In  the  following  table  are  given  some  results 
obtained  in  the  author's  laboratory  in  which  the  recognized  sources  of  error  have 
been  avoided  as  far  as  possible.  It  expresses  the  mean  values  obtained  in  the 
analysis  of  the  urine  of  six  well  nourished  men.  The  daily  excretion  is  taken  as 
1200  cc,  with  a  specific  gravity  of  1.023,  at  200  referred  to  water  at  40  as  1.000. 
In  grams  per  24  hours  we  have : 

Potassium,    K    2.82 

Sodium,   Na    4.87 

Calcium,  Ca   0.13 

Magnesium,   Mg    0.15 

Ammonium,  NH4  •  • 1.13 

Chlorine,  CI    •• 8.90 

Phosphoric   acid,    (P04) '"    2.41 

Sulphuric    acid,    (S04)" 2.73 

Urea,    CON2H4     3372 

Uric  acid,    (CBH2N403)"    0.88 

Creatinine,  C4H7N30    1.98 

.  Hippuric    acid,    (C9HSN03) '    1.00 

These  figures  are  merely  suggestive,  as  diet  makes,  naturally,  a  great  change  in 
the  excretion. 

Color.  In  health  the  straw-yellow  color  of  the  urine  is  characteristic,  the  depth 
of  shade  depending  largely  on  the  concentration.  With  the  same  solid  excretion 
in  24  hours  the  color  may  be  light  if  the  volume  of  water  consumed  is  large,  or 
it  may  be  a  deep  yellow  if  the  water  consumption  is  deficient.  These  facts  must 
be  kept  in  mind. 

Various  darker  shades  of  the  urine  may  be  observed  after  consumption  of  certain 
foods  or  certain  chemical  substances.  Rhubarb,  senna,  santonin,  salicylates  and  many 
other  aromatic  bodies  produce  highly  colored  urines.  In  some  cases  a  marked 
smoky  shade  is  observed,  and  this  is  usually  due  to  the  oxidation  of  more  or  less 
complex  phenols.  With  a  number  of  fruits  and  berries  a  bright  yellowish  or  yel- 
lowish-red color  is  noticed  in  the  urine. 

In  diseases  the  urine  may  be  colored  from  the  presence  of  substances  from  the 
blood,  the  bile,  or  from  absorbed  products  of  intestinal  putrefaction. 

Odor.  The  odor  of  urine  in  health  is  aromatic  and  absolutely  characteristic.  On 
standing  it  usually  changes  rapidly  from  the  action  of  bacteria,  and  then  an  am- 
moniacal  odor  is  ordinarily  developed,  through  the  alteration  of  the  urea.  Later, 
other  organic  matters  begin  to  break  down,  resulting  in  the  development  of  putre- 
factive or  other  disagreeable  odors. 


THE    URINE.  293 

Certain  remedies  impart  very  peculiar  odors  to  the  urine,  and  the  same  is  true 
of  several  vegetable  foods.  The  behavior  of  asparagus  and  turpentine  in  this  regard 
is  marked. 

In  disease  a  great  variety  of  organic  substances  may  be  carried  into  the  urine 
in  traces,  and  the  presence  of  these  is  often  accompanied  by  some  peculiar  odor. 
This  may  be  marked  enough  to  be  of  importance  in  diagnosis. 

Reaction.  The  urine  for  the  24  hours  is  normally  acid  to  litmus  paper.  This 
acidity  is  due  ordinarily,  to  the  presence  of  acid  salts,  rather  than  of  free  acid; 
among  the  acid  salts  the  di-hydrogen  sodium  phosphate  is  probably  the  most 
important. 

Under  normal  conditions  the  urine  may  become  temporarily  alkaline,  usually  from 
the  elimination  of  traces  of  alkali  carbonates  due  to  the  combustion  of  certain 
organic  salts  of  the  diet.  This  occasional  alkalinity  must  not  be  confounded  with 
that  which  is  very  commonly  observed  in  urine  which  has  been  passed  some  time. 
In  this  case  the  alkaline  reaction  is  due  to  the  presence  of  ammonium  carbonate 
coming  from  the  bacterial  decomposition  of  the  normal  urea. 

In  the  practical  examination  of  urine  litmus  papers  are  commonly  used  in  pref- 
erence to  other  indicators.  The  measurement  of  the  degree  of  acidity  is  uncertain. 
Occasionally  urine  shows  the  so-called  amphoteric  reaction ;  that  is,  it  turns  blue 
litmus  paper  red,  and  red  litmus  paper  blue.  Very  sensitive  paper  is  necessary 
to  show  this. 

The  Excretion  of  Alkali  Salts.  The  alkali  salts  found  in  the  urine 
come  from  the  sodium  chloride  consumed  as  such  in  salted  food,  and 
in  part  from  potassium  salts  in  the  juices  of  meat  and  in  vegetables. 
In  the  analysis  of  the  ash  of  muscle  given  some  pages  back  chlorine 
as  well  as  potassium  is  shown.  Chlorine  is  found,  although  usually  in 
small  amount,  in  the  ash  of  all  vegetable  substances.  In  the  latter, 
however,  especially  in  the  cereals,  potassium  phosphate  is  the  charac- 
teristic constituent  of  the  ash.  On  a  cereal  diet  we  should  expect  the 
urine,  in  consequence,  to  show  a  relatively  high  potash  and  phosphoric 
acid  content.  The  ash  of  potatoes  contains  in  the  mean  over  60  per 
cent  of  potassium  oxide  while  the  chlorine  is  in  excess  of  the  sodium. 
With  a  mixed  diet,  therefore,  the  composition  of  the  alkali  salts  in  the 
urine  must  be  variable  and  difficult  of  explanation.  As  the  alkali  com- 
pounds are  practically  all  soluble,  they  are  excreted  almost  solely  by 
the  urine  and  to  a  small  extent  only  by  the  feces.  The  analysis  of  the 
urine  gives  us  then,  in  ordinary  cases,  a  fairly  accurate  measure  of  the 
alkali  metals  taken  in  with  our  food  and  drink;  in  normal  condition 
there  is  no  accumulation  of  alkali  salts  in  the  body. 

Calcium  and  Magnesium  Compounds.  The  full  significance  of 
these  in  the  urine  we  can  not  explain,  since  without  complete  analyses 
of  the  feces  we  do  not  know  the  relation  of  the  excreted  to  the  ingested 
alkali-earths.  Our  natural  waters  contain  usually  appreciable  amounts 
of  these  salts,  with  those  of  calcium  in  excess  as  a  rule.  In  Lake 
Michigan  water,  for  example,  we  have  about  125  milligrams  per  liter 


294  PHYSIOLOGICAL    CHEMISTRY. 

of  these  salts  as  carbonates,  but  in  our  common  animal  and  vegetable 
foods  we  consume  daily  much  greater  quantities  than  we  could  get 
from  water.  The  ash  of  wheat  contains  about  12  per  cent  of  magnesia 
and  3  per  cent  of  lime,  while  in  the  ash  of  muscle  we  have  over  3  per 
cent  of  magnesia  and  between  2  and  3  per  cent  of  lime.  Five  hundred 
grams  of  lean  meat  would  furnish  us  then  with  over  150  milligrams 
of  magnesia  and  with  something  less  than  that  amount  of  lime. 

But  only  fractions  of  these  compounds  find  their  way  into  the  urine. 
In  the  original  foods  they  exist,  in  part  at  least,  in  insoluble  forms. 
While  some  of  these  substances  may  be  dissolved  in  the  stomach,  the 
conditions  are  reversed  in  the  intestines,  and  insoluble  phosphates,  car- 
bonates and  sulphates  are  lost  with  the  feces.  There  has  been  much 
discussion  as  to  the  exact  nature  of  the  calcium  and  magnesium  salts 
excreted.  In  a  measure  the  discussion  is  fruitless,  as  we  must  cer- 
tainly admit  the  free  exchange  of  ions  in  solution.  Under  ordinary 
conditions  the  acid  ions  of  the  urine  appear  to  be  in  slight  excess  of 
the  metals,  which  prevents  precipitation  of  insoluble  phosphates,  for 
example.  Temperature  plays  a  very  important  part  in  the  problem  of 
the  stability  of  the  calcium  and  magnesium  compounds  in  the  urine, 
and  the  problem  is  further  complicated  by  the  presence  of  uric  acid, 
the  peculiar  behavior  of  which  will  be  touched  upon  below. 

THE   NITROGEN    EXCRETION. 

For  many  reasons  this  excretion  is  the  most  important  which  we 
have  to  consider  in  connection  with  the  urine,  as  it  gives  us  an  insight 
into  some  of  the  fundamental  problems  in  metabolism.  The  largest 
part  of  it  leaves  the  body  as  urea,  but  the  proportion  excreted  in  other 
compounds  cannot  be  neglected.  We  have  pretty  accurate  methods 
for  the  estimation  of  urea,  ammonia,  uric  acid,  creatinine  and  purine 
nitrogen  as  they  are  found  in  the  urine.  Hippuric  acid,  which  is 
found  in  urine,  is  not  as  readily  measured,  and  for  several  other  com- 
pounds which  contain  nitrogen  our  methods  are  far  from  exact.  The 
following  table  shows  the  distribution  of  the  nitrogen  in  the  urine  of 
six  men  on  whose  complete  excretion  daily  tests  were  made  in  the 
author's  laboratory  through  a  period  of  four  months.  The  figures  are 
the  mean  values  for  the  whole  period,  and  are  in  percents  of  the  total 
nitrogen  excretion,  as  measured  by  the  Kjeldahl  process.  The  general 
mean  represents  720  determinations  for  each  constituent. 

Under  the  head  of  undetermined  nitrogen,  shown  in  the  table,  there 
is  included  the  nitrogen  of  hippuric  acid,  oxyproteic  acid,  alloxypro- 


THE    EXCRETION    OF    NITROGEN. 


295 


No. 

Urea 
Nitrogen. 

Ammonia 
Nitrogen. 

Purine 
Nitrogen. 

Uric  Acid 
Nitrogen. 

Creatinine 
Nitrogen. 

5.38 

5-52 
5-64 
5-50 
6.29 
4.94 

5-54 

Undetermined 
Nitrogen. 

I 
2 
3 

4 
5 
6 

83.26 
84.50 
82.43 
85.05 
81.46 
84.17 

4-39 
3.56 

5-55 
4-56 
4.71 
4.26 

4-50 

0.67 
0.6l 
O.36 
0.41 
0.6l 
0.51 

I.70 
I.69 
I.63 
1.23 

1.94 
I.69 

4.60 

4.12 

4-39 
3-25 
4.99 

4-43 

Mean. 

8348 

0.53 

1.65 

4.30 

teic  acid  and  traces  of  other  bodies  of  obscure  composition.     A  brief 
discussion  of  each  one  of  the  important  constituents  will  follow. 


UREA. 

The  relation  of  this  substance  to  ammonium  carbonate  has  been 
referred  to  many  times,  but  especially  in  discussing  the  enzymic  proc- 
esses of  the  liver.  The  nutrient  proteins  contain  many  amino  groups 
which  seem  to  be  split  off  in  the  general  combustion  or  hydrolytic 
processes  going  on  in  the  body;  also  a  great  excess  of  groups  which 
oxidize  more  completely  and  yield  carbon  dioxide.  The  large  part  of 
this  escapes  by  way  of  the  lungs,  while  another  part  is  evidently  taken 
care  of  in  the  liver  through  combination  with  the  amino  groups  to 
form  urea.  It  is  also  true  that  normally  some  of  this  amino  nitrogen 
fails  to  take  this  simple  course,  because  of  the  presence  of  strong  acid 
radicles,  which  have  great  tenacity  in  their  combining  reactions.  The 
ammonium  salts  so  formed  are  stable  and  cannot  be  worked  over 
into  urea. 

It  appears,  also,  that  the  nitrogen  of  some  other  groups  in  addition 
fails  to  reach  the  urea  stage.  Creatinine  and  uric  acid  nitrogen  are 
not  included  here,  as  these  substances  seem  to  have  an  independent 
origin  which  will  be  discussed  below.  But  there  are  obscure  com- 
pounds in  the  urine  in  small  amount  of  which  we  know  but  little,  and 
some  of  these  contain  nitrogen.  The  oxyprotcic  acid  referred  to  above 
is  an  illustration.  What  the  relation  of  this  is  to  urea  we  cannot  say, 
but  an  idea  of  this  kind  suggests  itself:  the  original  protein  complex 
may  contain  certain  groups  which  do  not  fall  an  easy  prey  to  the  work 
of  the  oxidation  enzymes  in  the  body;  they  do  not  break  down  to 
amino  compounds  and  carbon  dioxide,  but  remain  intact  as  very  resist- 
ant residues,  and  hence  when  the  liver  is  reached  they  are  not  in 
condition  to  pass  into  the  urea  stage.  In  the  katabolic  changes  of 
protein  it  is  possible  that  a  number  of  such  resistant  groups  may  be 
produced,  and  it  is  likely  that  the  amount  of  nitrogen  or  other  element 
which   so   escapes  the   normal   end    reaction   depends   largely  on   the 


296  PHYSIOLOGICAL    CHEMISTRY. 

strength  of  the  enzymic  functions.  These  must  vary  in  different  indi- 
viduals, and  hence  sometimes  more  and  sometimes  less  of  these  resist- 
ant, or  left  over,  residues  will  find  their  way  into  the  urine. 

From  this  point  of  view  urea  represents  that  part  of  the  original 
body  nitrogen,  aside  from  the  creatine  and  nuclein  derivatives,  which 
takes  the  normal  course.  It  represents  no  store  of  practically  realiz- 
able energy,  while  with  some  of  the  other  bodies  which  escape  in  the 
urine  this  is  not  the  case ;  under  more  favorable  conditions  they  might 
be  expected  to  suffer  further  oxidation  with  liberation  of  more  heat. 
Such  ideal  conditions  are  realized  in  some  individuals  more  than  in 
others. 

Urea  may  be  built  up  outside  of  the  body  by  many  synthetic  proc- 
esses, but  is  most  easily  prepared  by  the  conversion,  of  ammonium 
cyanate,  NH4OCN,  into  the  isomer.  On  evaporation  of  a  solution 
of  this  salt  the  transformation  into  urea  is  complete.  Urea  is  very 
soluble  in  water,  from  which  it  may  be  obtained  easily  in  crystalline 
form.  Its  solutions  are  easily  decomposed  by  many  oxidizing  agents 
with  formation  of  water,  carbon  dioxide  and  free  nitrogen,  on  which 
behavior  several  of  the  processes  for  determining  it  are  based.  This 
change  is  brought  about  by  hypochlorites,  for  example,  in  this  manner : 

CON.H*  +  3NaOCl  =  3NaCl  +  2H20  +  C02  +  N2. 

The  amino  groups  in  urea  may  be  completely  converted  into  ammo- 
nia in  many  ways,  and  this  reaction,  also,  is  applied  in  estimating  urea, 
as  will  be  shown  in  the  next  chapter. 

On  the  other  hand,  urea  may  take  part  in  synthetic  reactions  and 
may  be  combined  to  form  complex  substances,  in  certain  cases,  as  will 
be  shown  below. 

AMMONIA. 

This  represents  a  portion  of  the  protein  disintegration  which  for  a 
number  of  reasons  has  not  been  converted  into  urea.  The  ammonia 
passing  into  the  urine  takes  that  course  ordinarily  through  combination 
with  mineral  or  other  acids,  which  are  not  destroyed,  or  may  not  be 
destroyed,  by  oxidation.  In  any  pathological  increase  of  such  acids, 
if  there  is  not  enough  fixed  alkali  in  the  blood  to  combine  wifh  them, 
ammonia  is  split  off  from  protein  derivatives  in  quantity  sufficient  to 
complete  the  neutralization.  This  may  be  shown  also  by  the  injection 
of  free  mineral  acids  either  directly  or  with  the  food;  an  increased 
elimination  of  ammonia  results.  It  should  be  expected,  therefore,  that 
the  proportion  of  ammonia  in  the  urine  would  be  subject  to  marked 
fluctuations,  which  is  indeed  the  case.     Taken  with  other  determina- 


THE    EXCRETION    OF    NITROGEN.  297 

tions  the  estimation  of  ammonia  may  possess  considerable  diagnostic 
value,  as  it  measures  to  some  extent  the  excessive  acid  excretion.  In 
advanced  stages  of  diabetes,  with  marked  elimination  of  acid,  the 
ammonia  content  of  the  urine  may  increase  to  several  grams  daily. 
The  normal  amount  is  usually  a  gram  or  less. 

Ammonia  must  be  determined  in  fresh  urine  only,  since  in  old  urine 
fermentation  changes  soon  produce  large  quantities  of  the  substance 
from  the  breaking  down  of  urea. 

URIC  ACID  AND  THE  PURINE  BODIES. 

Few  topics  in  physiological  chemistry  have  attracted  more  attention 
than  the  relations  of  uric  acid  to  other  nitrogenous  products  excreted 
in  the  urine,  and  its  behavior  in  relation  to  disease.  The  importance 
of  the  substance  in  this  point  of  view  has  undoubtedly  been  very  fre- 
quently over-estimated  and  even  at  the  present  time  clinicians  are  much 
divided  as  to  the  part  it  plays  in  certain  diseases.  This  much  may  be 
said  with  truth,  however,  that  many  of  the  fine-spun  theories  which 
have  been  advanced  by  medical  men  on  the  uric  acid  question,  and 
which  have  held  our  attention  for  a  longer  or  shorter  period,  have 
been  founded  on  very  weak  chemical  evidence,  and  this,  it  should  be 
mentioned,  is  the  real  factor  in  the  case. 

Under  the  older  view,  as  explained  already,  uric  acid  was  supposed 
to  be  but  a  step  in  the  formation  of  urea,  the  normal  end  product  in 
protein  metabolism,  and  numerous  disorders  were  attributed  to  the 
accumulation  of  uric  acid  in  the  blood  through  some  failure  in  the 
final  oxidation  processes.  But  it  appears  now  from  the  evidence  avail- 
able that  uric  acid  is  not  a  natural  step  in  the  oxidation  of  the  simple 
proteins;  it  does  result,  however,  from  the  breaking  down  of  the 
complex  nucleo-proteids  which  are  represented  to  a  limited  extent  only 
in  the  body,  as  compared  with  the  muscle  proteins,  for  example.  The 
glandular  organs  rich  in  cells  furnish  the  chief  amount  of  the  nuclein 
complexes.  In  the  katabolism  of  these,  true  proteins  and  the  residues 
rich  in  phosphorus  known  as  nucleic  acids  result ;  the  proteins  undergo 
the  usual  further  oxidation  probably,  while  the  nucleic  acids  break 
down  into  a  variety  of  products  of  which  the  purine  bases,  the  pyrimi- 
dine  bases,  phosphoric  acid  and  carbohydrate  groups  are  the  most  im- 
portant. The  purine  bodies  in  turn  doubtless  give  rise  to  uric  acid. 
As  pointed  out  in  Chapter  V  several  nucleic  acids  exist;  their  struc- 
tural formulas  are  not  known,  but  empirically  these  formulas  have  been 
given  to  acids  from  different  sources. 


298  PHYSIOLOGICAL    CHEMISTRY. 

C40H52N14O:,5P4    Salmon  milt 

C40H5eN14O26P4 Salmon  milt 

C36H4SN14O30P4    Yeast  cells 

C41H61N16031P4   Wheat  embryo 

The  cleavage  products  of  these  acids  are  not  constant,  since  from  dif- 
ferent acids  different  purine  bases  have  been  made.  Those  found  in 
the  animal  body  are  the  following:  xanthine,  hypoxanthine,  guanine, 
adenine,  heteroxanthine,  paraxanthine  and  epiguanine.  In  order  to 
show  the  relations  of  these  compounds  to  uric  acid,  E.  Fischer  pro- 
posed to  consider  them  all  as  derivatives  of  a  nucleus  group  which  he 
called  purine. 

As  the  chemistry  of  these  bodies  is  complex  it  may  be  well  to  illus- 
trate their  relations  by  the  structural  formulas  worked  out  or  con- 
firmed by  Fischer.  Starting  with  the  assumed  purine  nucleus  we  have 
these  formulas,  with  the  nucleus  atoms  numbered,  as  suggested  by 
Fischer : 

T  n — C  6  N=CH  HN— CO 

I         I      7  I       I      H  || 

2  C  5  C— Nv  HC    C— Nv  OC    C— NH 

I         I         >C8  ||      ||        )CH  ;       !!        \ 

3  n — C— W  N— C— N^  ^CH 

4      9  HN-C-N/ 

Purine  nucleus,  C5N4  Purine,  CEH4N4  Xanthine,  CsH^NiOa 

HN— CO  N=C— NH2  HN— CO 

OC— C— NH  HC    C— N  HC    C— NH 

)C0  jl      I        ^CH  ||      I        ^CH 

HN— C— NH  N— C— N— H  N— C— N 

Uric  acid,  C5H4N403  Adenine,  C5H5N6  Hypoxanthine,  CbILN^O 

Employing  the  Fischer  nomenclature  these  bodies  have  the  follow- 
ing names : 

Adenine 6-aminopurine 

Hypoxanthine     6-oxypurine 

Xanthine 2,  6-dioxypurine 

Uric  acid   2,  6,  8-trioxypurine 

Guanine    2-amino-6-oxypurine 

As  their  relations  have  been  shown  by  various  syntheses  and  other 
transformations,  and  as  further,  the  xanthine  and  hypoxanthine,  ade- 
nine and  guanine  have  been  directly  derived  from  the  nucleic  acids, 
the  relation  of  uric  acid  to  the  latter  bodies  is  not  far  to  seek. 

Not  all  of  the  nucleic  acid  destroyed  can  be  assumed  to  come  from 
body  cell  structures;  many  of  our  foods  contain  nucleins  and  these 
must  give  rise  to  the  same  derivatives  on  oxidation  without  passing 


THE    EXCRETION    OF    NITROGEN.  299 

through,  becoming  part  of,  the  cells  of  the  glandular  organs  of  the 
body.  Accordingly  we  distinguish  between  endogenous  and  exoge- 
nous purines  and  uric  acid.  With  the  food  nucleins  eliminated  as  far 
as  possible,  it  has  been  found  that  the  uric  acid  excreted  becomes  nearly 
constant  and  bears  a  more  uniform  relation  to  the  urea.  This  indi- 
cates that  the  destruction  of  cell  substance  in  the  body  leads  as  regu- 
larly to  uric  acid  as  does  that  of  muscle  proteins  to  urea.  The  use  of 
rich  protein  foods  does  not  necessarily  occasion  greater  elimination  of 
uric  acid.  It  is  only  when  they  contain  appreciable  amounts  of  the 
nucleins  that  this  is  the  case.  In  addition  to  these  facts  it  has  been 
found  experimentally  that  the  oxidation  of  nucleins  outside  the  body 
leads  to  the  production  of  uric  acid  in  small  amount. 

Uric  acid  may  be  obtained  synthetically  by  combining  urea  with 
glycocoll,  and  at  a  high  temperature  it  may  be  decomposed  with  pro- 
duction of  urea,  ammonia,  prussic  acid  and  other  bodies,  under  dif- 
ferent conditions.  But  little  importance  is  attached  to  these  facts  at 
the  present  time,  but  formerly  they  were  supposed  to  support  the  view 
that  uric  acid  is  a  stage  in  the  urea  formation  through  which  all  the 
katabolic  nitrogen  should  pass.  Of  greater  interest  is  this  fact  that 
when  uric  acid  is  introduced  into  the  circulation  of  certain  animals 
some  of  it  appears  to  be  destroyed,  and  with  the  production  of  a  little 
urea.  Such  observations  suggest  that  possibly  a  small  part  of  our 
urea  may  come  from  uric  acid,  but  they  have  no  bearing  on  the  propo- 
sition that  the  acid  in  turn  has  its  origin  in  the  nucleins  and  not  in 
the  common  proteins. 

According  to  the  structural  formula  above  given  uric  acid  appears 
to  have  four  hydrogen  atoms  of  equal  value  in  the  formation  of  salts. 
But  apparently  only  two  classes  of  salts  may  be  formed :  neutral  salts, 
in  which  two  hydrogens  are  replaced,  and  acid  salts,  in  which  but 
one  hydrogen  is  replaced.  We  have  therefore  salts  of  the  types 
MC5H3N403  and  M2C5H2N403.  In  addition  to  these,  so-called 
quadrinrates  are  known  as  urine  sediments.  These  salts  are  of  the 
type  MC5H3N403C5H4N403.  The  pure  acid  requires  nearly  40,000 
parts  of  water  for  solution;  the  neutral  salts  of  the  alkali  metals  are 
much  more  soluble ;  while  the  acid  salts  are  but  slightly  soluble.  The 
data  given  by  different  observers  are  very  contradictory.  The  salts 
of  barium,  strontium  and  magnesium  are  nearly  insoluble  in  water. 

Solutions  of  urates  in  presence  of  alkali  exhibit  a  reducing  action 
toward  copper,  silver  and  certain  other  salts,  which  fact  possesses  an 
importance  which  will  be  explained  later. 


3<X)  PHYSIOLOGICAL    CHEMISTRY. 

XANTHINE  AND  THE  OTHER  PURINES. 

The  amount  of  these  purines  not  changed  to  uric  acid  is  small,  the  sum  of  all  of 
the  nitrogen  so  held  not  being  over  one-third  of  the  uric  acid  nitrogen,  according 
to  most  observers.  They  are  but  slightly  soluble  in  pure  water,  but  dissolve  readily 
with  alkali  hydroxides  to  form  compounds  like  the  urates.  With  the  heavy  metals 
the  combinations  are  mostly  insoluble. 

PYRIMIDINE  DERIVATIVES. 

Among  the  decomposition  products  of  nucleic  acids  the  so-called  pyrimidines 
should  be  referred  to,  as  they  must  bear  some  relation  to  the  urinary  nitrogen. 
Pyrimidine  itself  represents  a  nucleus  like  purine  from  which  various  derivatives 
may  be  formed.     The  relations  as  outlined  by  Kossel  are  these: 

i  N=C— H  6  NH— C=0  NH— CO 

II  II  II 

2  CH  C— H  s  C=0  C— H  C=0  C— CH3 

II      II  I  II  II 

3  N— C— H  4  NH— C— H  NH— CH 

Pyrimidine,  C4H4N2  Uracil,  C4H4N202  Thymine,  C5H6N202 

2,  6-dioxy  pyrimidine  5-methyl  uracil 

We  have  no  definite  knowledge  of  the  occurrence  of  these  bodies  in  urine,  but 
they  are  among  the  compounds  which  should  be  expected  to  result  from  the  nuclein 
metabolism  and  possibly  form  a  part  of  the  nitrogen  residue  not  fully  accounted 
for.  Thymine  has  been  obtained  as  a  well-crystallized  compound,  soluble  readily  in 
warm  water.     It  is  identical  with  the  body  formerly  described  as  nucleosin. 

CREATININE. 

Creatinine,  C4H7NsO,  is  the  anhydride  of  the  creatine  described 
under  the  head  of  the  muscle  extractives,  and  is  always  present  in 
normal  urine.  The  amount  in  which  it  occurs  is  indicated  by  the 
analyses  given  above.  Much  has  been  written  on  the  question  of  the 
relation  of  creatine  to  urea,  but  the  evidence  for  the  view  held  by  some 
writers  that  the  latter  is  derived  from  the  former  is  not  very  con- 
vincing. In  a  laboratory  way  by  boiling  creatine  with  baryta  water, 
urea,  sarkosine  and  several  other  things  result.  But  no  corresponding 
reaction  appears  to  take  place  in  transfusion  experiments  with  crea- 
tine, and  it  has  been  held  until  recently  that  creatine  introduced  with 
the  food  appears  in  the  urine,  not  as  urea,  but  as  creatinine. 

Of  this  transformation  there  is  now  considerable  doubt,  and  we  have 
at  present  no  very  satisfactory  theory  concerning  the  origin  of  the 
urinary  creatinine.  Much  of  our  information  on  this  subject  has  come 
from  the  investigations  of  Folin,  who  pointed  out  a  few  years  ago  that 
the  elimination  of  creatinine  is  fairly  constant  for  any  given  individual 
from  day  to  day,  but  varies  in  different  individuals.  The  total  elimi- 
nation seems  to  be  independent  of  muscular  exertion,  and  varies  with 
the  body  weight,  amounting  to  about  16  to  20  milligrams  per  kilo 
daily.     The  excretion  is  not  essentially  changed  by  a  high  protein 


THE    EXCRETION    OF    NITROGEN.  30 1 

diet,  and  is  not  lowered  by  fasting.  It  may  be,  then,  the  measure  of 
some  peculiar  phase  of  nitrogenous  metabolism  characteristic  for  each 
individual. 

The  conversion  of  creatine  into  creatinine  by  boiling  with  weak 
acids  follows  quantitatively  according  to  the  following  reaction,  but 

the  change  is  not  rapid,  as  formerly  assumed. 

NH 
yNH2  /  \ 

\T  CHS  -»        H-N=C\M  CH2  •  CO  +  H20 

NCH2-COOH  iNCH3 

But  in  alkaline  solution  the  reverse  reaction  takes  place,  creatine  being 
slowly  formed.  Creatinine  may  be  separated  from  the  urine  most 
easily  with  zinc  chloride  as  a  crystalline  double  salt  which  is  yellow 
because  of  the  coprecipitation  of  pigments;  when  purified  the  crystals 
are  colorless.  Creatinine  is  found  in  normal  amount  in  the  urine  of 
vegetarians.  Toward  copper  and  some  other  metallic  solutions  it  be- 
haves as  a  reducing  agent  when  alkali  is  present. 

OTHER  NITROGENOUS  BODIES. 

It  has  been  intimated  that  other  compounds  of  nitrogen,  besides 
those  just  mentioned  are  excreted  with  the  urine.  Of  the  nature  and 
amount  of  these  much  remains  to  be  shown  by  experiment.  It  was 
pointed  out  that  4  to  5  per  cent  of  the  total  urine  nitrogen  in  the  table 
given  above  was  "undetermined."  It  did  not  belong  to  the  urea, 
uric  acid  or  other  purines,  ammonia  or  creatinine.  Among  the  bodies 
which  contain  some  of  this  nitrogen  the  following  may  be  mentioned 
as  the  most  important. 

Hippuric  Acid.  This  is  benzoyl  glycine,  C6H5CO  •  NHCH2COOH, 
and  is  always  present  in  some  amount  in  normal  urine.  It  is  formed 
by  synthesis  from  the  glycine  group  of  metabolism,  and  the  benzoic 
acid  ingested  with  many  aromatic  food  substances,  certain  acid  fruits 
and  spices,  for  example.  Some  aromatic  products  of  protein  metabo- 
lism may  also  give  rise  to  it.  Moderate  doses  of  benzoates,  or  benzoic 
acid,  appear  in  the  urine  wholly  in  the  form  of  hippuric  acid,  as  the 
glycine  groups  seem  to  be  always  present  in  sufficient  amount  to  com- 
plete the  synthesis.  These  groups,  otherwise,  go  to  form  urea,  appar- 
ently. The  hippuric  acid  content  of  the  urine  is  far  from  constant,  as 
it  depends  so  largely  on  the  diet,  but  a  gram  or  more  daily  is  often 
present.     The  nitrogen  in  the  acid  amounts  to  7.8  per  cent. 

Oxyproteic  Acid.  This  product  was  first  described  by  Bondzynski 
and  Gottlieb,  and  has  been  studied  by  others.     The  exact  formula  is 


302  PHYSIOLOGICAL    CHEMISTRY. 

not  known,  but  the  percentage  composition  is  about  C,  39.62;  H,  5.64; 
N,  18.08;  S,  1. 12;  O,  35.54.  Under  the  names  of  antoxyproteic  acid 
and  alloxyproteic  acid  Bondzynski  and  other  colleagues  have  described 
somewhat  related  substances.  As  these,  and  still  other  bodies  which 
have  been  found  in  urine  contain  both  sulphur  and  nitrogen,  it  is  evi- 
'dent  that  they  represent  a  peculiar  small  fraction  of  the  original  protein 
which  has  failed  to  be  hydrolyzed  and  oxidized  in  the  general  metabo- 
lism. It  has  been  estimated  that  2  or  3  per  cent  of  the  total  urine 
nitrogen  is  found  in  these  compounds,  and  the  sulphur  in  them  would 
be  included  in  the  so-called  "  neutral "  sulphur,  to  be  referred  to  later. 
Pathologically  the  relations  in  the  nitrogen  excretion  may  be  very 
much  changed,  and  protein,  as  such,  may  occur  in  the  urine  in  con- 
siderable amount.  The  detection  of  this  protein  will  be  discussed  in 
the  following  chapter,  along  with  the  methods  for  the  measurement  of 
the  normal  constituents. 

THE  SULPHUR  EXCRETION. 

The  sulphur  in  the  urine  is  found  in  several  compounds  and  comes 
from  different  sources.  A  small  part  has  its  origin  in  traces  of  sul- 
phates taken  directly  in  food  and  natural  waters;  some  comes  from 
the  sulphur  existing  in  peculiar  combinations  in  certain  vegetables, 
while  the  largest  part  has  its  origin  in  the  sulphur  of  proteins,  which 
undergoes  more  or  less  complete  oxidation  before  elimination  through 
the  kidneys.  Most  of  this  sulphuric  acid  of  oxidation  combines  with 
alkalies  for  elimination;  if  fixed  alkalies  are  deficient  ammonium  sul- 
phate is  formed  and  this  ammonia  therefore  escapes  the  natural  oxida- 
tion to  urea. 

It  has  been  explained  in  an  earlier  chapter  that  in  the  putrefactive 
changes  taking  place  in  the  intestines  certain  phenol  bodies  are  split 
off  from  proteins  there  remaining,  or  perhaps  in  most  cases  from  the 
unabsorbed  protein  derivatives.  A  very  considerable  part  of  these 
phenol  bodies  escapes  with  the  feces,  but  another  portion,  often  much 
increased  in  disease,  is  absorbed  by  the  blood  vessels  from  the  lower 
intestine  and  carried  to  the  liver  where  combination  with  sulphuric 
acid  is  effected,  probably  through  some  kind  of  enzymic  action.  From 
recent  observations  it  appears  likely  that  the  combination  is  effected 
before  the  sulphur  is  completely  oxidized,  that  is,  while  it  is  in  the 
sulphite  condition.  The  final  oxidation  then  follows.  The  ethereal 
sulphate  so  formed  is  discharged  finally  with  the  urine.  The  fraction 
of  the  sulphuric  acid  so  voided  is  extremely  variable,  reaching  some- 
times 20  per  cent  of  the  whole.     The  most  abundant  of  these  combi- 


THE   EXCRETION    OF    SULPHUR. 


303 


nations  are  salts  of  phenyl-,  cresyl-,  indoxyl-,  and  skatoxyl-sulphuric 

acid,  the  structural  relations  of  which  are  shown  by  the  following 

formulas : 

CH 

//\ 
HC      C — C.O.SCUDH 


CeH50  -1  c;o       CH3C„H40  \  qo 

r  bU=  •  HO  /  bU= 


HO 


Phenyl-sulphuric  acid     Cresyl-sulphuric  acid 


HC      C      CH 

\/\/ 

C        N 
H       H 

Indoxyl-sulphuric  acid 


Skatoxyl-sulphuric  acid  is  the  methyl  derivative  of  indoxyl-sulphuric 
acid.  Phenyl-  and  cresyl-sulphuric  acids  are  frequently  called  phenol- 
and  cresol-sulphuric  acids,  but  the  former  names  are  preferable.  The 
alkali  salts  of  indoxyl-sulphuric  acid  are  known  as  indican,  the  appear- 
ance of  which  in  the  urine  in  quantities  above  minute  traces  is  an  indi- 
cation of  the  existence  of  excessive  putrefactive  reactions  in  the  intes- 
tines. The  observation  is  therefore  of  value  in  diagnosis.  Accurate 
methods  are  known  for  the  recognition  of  the  substance  in  the  urine. 

In  addition  to  the  oxidized  or  sulphate  sulphur  in  the  urine  there  are 
always  traces  of  so-called  "  neutral "  sulphur  compounds  present.  Ac- 
cording to  various  authorities  the  sulphur  in  these  compounds,  which 
are  all  complex  organic  bodies,  may  make  up  12  to  25  per  cent  of  the 
total  sulphur.  The  sulphur  bodies  here  in  question  are  not  easily 
recognized  in  most  cases  and  the  existence  of  several  of  them  described 
in  the  journals  is  yet  to  be  demonstrated.  The  fact  of  the  excess  of 
sulphur  over  that  contained  in  the  mineral  and  ethereal  sulphates  may 
be  easily  shown  by  determination  of  the  total  sulphates  directly,  and 
then  after  complete  oxidation  of  the  urine  by  sodium  peroxide,  or 
other  agent.  This  unoxidized  or  neutral  sulphur  is  found  in  such 
bodies  as  the  oxyproteic  acid  mentioned  above,  and  occasionally  in 
cystin  or  taurin  present. 

The  following  table  gives  a  good  idea  of  the  sulphur  excretion 


No. 

Inorganic  Sulphur. 

Ethereal  Sulphur. 

Neutral  Sulphur. 

I 

72.30 

9.71 

17.98 

2 

74.96 

9.42 

15.62 

3 

75-93 

8.48 

15  59 

4 

78.13 

6-55 

I5-32 

5 

71.84 

9-13 

1903 

6 

76.80 

6.39 

16.81 

Mean. 

74-99 

8.28 

'6.73 

through  a  long  period,  in  the  urine  of  six  men  in  normal  health.     The 
values  represent  fractions  of  the  total  sulphur,  and  in  each  case  are  the 


304  PHYSIOLOGICAL    CHEMISTRY. 

means  of  daily  observations  on  the  whole  urine,  through  four  months. 
The  observations  were  made  in  the  author's  laboratory. 

THE  PHOSPHORUS  EXCRETION. 

As  mentioned  in  an  earlier  chapter,  some  of  our  foods  are  very  rich 
in  phosphates,  or  phosphate-furnishing  material.  This  is  especially 
true  of  the  cereals,  of  a  few  animal  proteins  and  of  the  lecithins.  The 
phosphates  formed  by  oxidation  of  these  substances  are  mainly  elimi- 
nated with  the  urine,  and  in  traces  with  the  feces.  A  very  consider- 
able portion  of  the  urinary  phosphoric  acid  comes  from  this  source, 
which  may  be  described  as  the  exogenous  phosphoric  acid.  Another 
portion  comes  from  the  breaking  down  of  the  nucleic  acids  of  the  cell 
substances  and  from  the  so-called  phospho-globulins  or  nucleo-albumins 
also  found  in  the  body.  These  are  the  endogenous  sources.  How  the 
phosphorus  is  held  in  these  last-named  bodies  is  not  known,  but  in  the 
nucleic  acids  it  appears  to  be  in  oxidized  form,  according  to  some 
authorities  as  metaphosphoric  acid.  The  excreted  product  is  ortho- 
phosphoric  acid,  combined  to  form  salts  of  the  type  MH2P04  or 
M2HP04.  The  alkali  salts  are  soluble  readily,  while  those  of  cal- 
cium and  magnesium  are  only  in  part  soluble  in  water.  The  condi- 
tions of  solubility  in  urine  are  complicated  by  the  presence  of  other 
salts. 

The  amount  of  phosphoric  acid,  as  P205,  excreted  daily  varies 
within  wide  limits;  according  to  the  above  analysis  the  mean  may  be 
about  2.5  gm.  If  the  urine  becomes  alkaline  through  the  fermenta- 
tion of  urea  a  very  considerable  part  of  the  phosphate  may  be  precipi- 
tated in  the  form  of  calcium  and  magnesium  salts.  One  of  the  com- 
monest of  these  is  the  so-called  triple  phosphate,  NH4MgP04-6H20. 
The  determination  of  the  amount  of  phosphoric  acid  in  the  urine  will 
be  discussed  in  the  next  chapter. 

CARBOHYDRATES. 

Normally  no  large  amount  of  any  carbohydrate  passes  from  the 
blood  into  the  urine,  but  with  increased  sugar  concentration  in  the 
blood  from  any  cause,  the  urine  may  contain  sugar  in  more  than  traces. 
When  sugars  are  consumed  in  more  than  the  "assimilation  limit"  a 
part  of  the  excess  may  be  found  in  the  urine.  This  is  true  of  glucose 
as  well  as  of  cane  sugar  and  maltose. 

But  under  normal  conditions  there  is  evidence  that  traces  of  several 
carbohydrates  and  of  bodies  related  to  them  pass  also  into  the  urine. 
It  is  well  known  that  the  oc-naphthol  reaction  is  given  with  ordinary 


THE    EXCRETION    OF    CARBOHYDRATES    AND    PROTEINS.  305 

normal  urines,  and  this  points  to  some  carbohydrate  derivative  which 
can  yield  furfuraldehyde.  The  other  delicate  sugar  tests  give  the 
same  indication.  Some  of  the  carbohydrate  doubtless  appears  as  such, 
but  a  portion  of  it  probably  exists  in  the  form  of  glucoproteids  or 
other  complex  groups.  Part  of  the  carbohydrate  can  be  readily  fer- 
mented, while  another  portion  resists  the  action  of  yeast.  Such 
observations  may  be  interpreted  as  suggesting  the  presence  of  other 
carbohydrates  than  the  common  glucose.  More  will  be  said  about 
this  below. 

Pathologically,  the  passage  of  even  large  quantities  of  sugar  into 
the  urine  is  a  common  phenomenon.  This  is  most  frequently  observed 
in  diabetes  mellitus,  a  disease  in  which  the  power  of  oxidizing  sugar 
in  the  muscles  seems  to  be  wholly  or  partly  lost.  It  has  been  already 
mentioned  that  this  oxidation  is  possibly  an  enzymic  operation  in  which 
certain  muscle  and  pancreas  enzymes  are  at  the  same  time  active.  In 
this  form  of  diabetes  several  hundred  grams  daily  of  sugar  may  be 
excreted. 

In  another,  "  artificial,"  form  of  diabetes  sugar  may  be  caused  to 
appear  in  considerable  quantity  temporarily  in  the  urine.  The  admin- 
istration of  small  doses  of  phloridzin,  a  glucoside,  is  followed  by  the 
appearance  of  sugar  in  the  urine,  but  this  condition  is  now  believed  to 
depend  on  a  disturbance  of  the  proper  functions  of  the  kidneys  rather 
than  on  any  alteration  of  the  sugar-oxidizing  power.  The  power  of 
retaining  the  sugar  in  the  blood  from  which  it  should  be  taken  up  and 
oxidized  by  the  muscular  tissues  depends  on  the  maintenance  of  the 
integrity  of  the  membranous  structures  of  the  kidneys.  If  these  suffer 
strain,  which  seems  to  be  the  case  after  administration  of  phloridzin, 
a  little  sugar  more  than  the  normal  may  be  able  to  diffuse  or  pass 
through  in  some  manner.  It  is  possible  that  many  other  substances 
may  have  a  somewhat  similar  action,  but  in  much  smaller  degree,  and 
so  occasion  sugar  excretions  still  within  what  may  be  called  "  normal " 
limits. 

PROTEINS. 

As  in  the  case  of  the  sugars,  so  with  the  proteins;  there  is  good  evi- 
dence that  traces  of  these  nitrogen  compounds  are  normally  present  in 
the  urine.  The  amount  which  may  be  present  is,  however,  very  small, 
in  the  mean  not  over  40  to  50  milligrams  per  liter.  With  these  traces 
it  is  not  possible  to  say  how  many  different  kinds  of  proteins  may 
occur,  but  the  serum  albumin  is  doubtless  the  most  abundant.  With 
greatly  increased  amounts  of  albumin  in  the  diet  the  excreted  albumin 
is  also  increased,  with  no  visible  impairment  of  the  kidney  mechanism. 


306  PHYSIOLOGICAL    CHEMISTRY. 

It  is  also  true  that  foreign  albumins  injected  into  the  blood  circulation, 
white  of  egg,  for  example,  are  speedily  eliminated  by  the  kidneys. 

Pathologically,  however,  the  serum  albumin  and  serum  globulin  of 
the  blood  may  pass  through  the  kidneys  in  considerable  quantity. 
This  is  occasionally  due  to  disturbances  in  the  circulation  which  may 
bring  about  an  increase  of  blood  pressure,  but  ordinarily  is  due  to 
structural  changes  in  the  kidneys  themselves,  through  which  the  power 
of  perfectly  retaining  albumins  of  the  blood  is  lost.  A  discussion  of 
the  nature  of  these  changes  is  not  within  the  scope  of  this  book,  and 
for  an  explanation  of  the  tests  which  are  employed  in  recognizing  the 
proteins  of  the  urine  the  reader  is  referred  to  the  next  chapter.  Many 
of  these  tests  are  but  modifications  of  the  delicate  protein  tests  de- 
scribed in  one  of  the  earlier  chapters. 

URINARY  SEDIMENTS. 

The  urine  when  passed  is  usually  clear,  but  frequently  it  soon  be- 
comes cloudy  and  deposits  a  precipitate.  This  precipitate  may  consist 
of  a  variety  of  constituents  and  may  be  due  to  several  causes.  Sooner 
or  later  all  urines,  unless  specially  protected  by  preservatives,  undergo 
the  so-called  ammoniacal  fermentation  in  which  urea  is  converted  into 
ammonium  carbonate  by  certain  bacteria.  When  this  alkaline  condi- 
tion is  reached  the  condition  of  equilibrium  in  which  the  various  salts 
exist  together  is  destroyed  and  insoluble  products  are  commonly 
formed  which  appear  as  precipitates.  The  alkali-earth  phosphates  are 
the  bodies  usually  thrown  out  in  this  way. 

But  disturbances  in  the  equilibrium  may  result  from  changes  of 
temperature  also,  and  precipitation  occur  because  of  the  simple  cooling 
of  the  warm  voided  urine.  Many  of  the  peculiar  urate  sediments  are 
formed  in  this  way.  Sometimes  the  separation  of  sediment  begins  in 
the  bladder  or  ureters  and  this  sediment  may  take  the  form  of  hard 
concretions  or  calculi.  In  years  past  numerous  attempts  have  been 
made  to  explain  the  formation  of  these  deposits,  especially  as  they 
occur  within  the  body.  The  theoretical  explanations  given  have  been 
in  general  far  from  satisfactory,  and  the  most  recent  studies  have  only 
gone  to  show  the  complexity  of  the  problem.  It  is  now  coming  to  be 
recognized  that  we  have  here  one  of  the  most  difficult  problems  of 
physical  chemistry,  which  like  other  questions  of  chemical  equilibrium 
in  solution  may  be  approached  only  through  elaborate  studies.  The 
beginning  of  such  studies  may  be  seen  in  some  of  the  valuable  papers 
which  have  been  published  in  the  last  few  years  on  the  solubility  of 
uric  acid  and  its  salts,  and  the  degrees  of  dissociation  which  obtain 


REDUCING    POWER    OF    NORMAL    URINE.  3O7 

in  the  various  solutions.     As  yet  these  matters  are  scarcely  in  condition 
for  elementary  presentation. 

The  recognition  of  the  general  character  of  the  sediments  from 
urine  is  most  readily  effected  by  the  aid  of  the  microscope,  which  is 
explained  in  the  next  chapter. 

THE  REDUCING  POWER  OF  NORMAL  URINE. 

On  account  of  the  presence  of  some  of  the  bodies  described  in  the 
last  few  pages,  normal  urine  exhibits  a  certain  reducing  action  toward 
metallic  solutions.  At  one  time  this  was  ascribed  to  traces  of  carbo- 
hydrates present,  but  later  doubt  was  thrown  on  this  conclusion  and 
the  presence  of  even  traces  of  sugar-like  bodies  was  denied  by  most 
writers  concerned  with  the  question.  With  the  development  of 
greater  accuracy  in  the  methods  of  detecting  sugars,  especially  through 
the  aid  of  the  phenylhydrazine  combination  and  the  formation  of  ben- 
zoic esters,  the  question  of  the  passage  of  traces  of  carbohydrates  into 
the  urine  seems  to  be  finally  settled  in  the  affirmative,  but  the  amount 
of  sugar  which  may  be  so  identified  is  too  small  to  account  for  the 
total  reduction  easily  measured  by  copper  solutions.  This  normal 
reduction  cannot  be  quantitatively  followed  with  the  ordinary  Fehling 
solution,  but  if  a  dilute  Pavy  solution,  as  described  in  Chapter  III,  is 
used,  a  very  satisfactory  determination  may  be  made.  This  modified 
Pavy  solution  is  given  such  a  strength  that  I  cc.  oxidizes  i  mg.  of 
glucose  in  very  dilute  solution,  approximating  0.2  per  cent  strength 
or  less. 

In  a  large  number  of  tests  made  in  the  author's  laboratory  a  few 
years  ago  it  was  found  that  to  reduce  50  cc.  of  such  a  solution  urine 
volumes  ranging  between  14.9  cc.  and  58  cc.  were  required.  The 
mean  of  all  the  determinations  on  these  normal  urines  was  23  cc.  In 
hundreds  of  normal  urines  examined  since  similar  results  have  been 
obtained.  In  all  these  urines  the  creatinine  and  uric  acid  present  were 
accurately  determined  and  an  attempt  was  made  to  connect  these  bodies 
with  the  reduction. 

The  Reducing  Power  of  Creatinine.  The  reducing  power  of  creatinine  may  be 
easily  found  by  the  method  referred  to,  using  the  weak  ammoniacal  copper  solution. 
A  liter  of  this  solution  contains  8.166  gm.  of  CuS04-5H20,  corresponding  to  2.6042 
gm.  of  CuO.  A  measured  volume  of  this,  usually  25  or  50  cc,  is  reduced  by  a 
creatinine  solution  of  definite  strength  and  the  volume  required  noted.  The  details 
me  experimenti  are  «ivcn  in  the  following  tabic.  The  copper  solution  was 
diluted  to  100  re.  before  the  titration: 


3o8 


PHYSIOLOGICAL    CHEMISTRY. 


Creatinine 
in  ioo  cc. 

Copper  Solution 
Taken 

CuO   Equivalent. 

Creatinine  Solu- 
tion Used. 

Creatinine  to 
130.2  mg.  CuO. 

Mols.  CuO 

to  1  Mol. 

C4H7N30. 

50  mg. 

50 
I20 
I20 

25  CC. 
25 

50 

65.I  mg. 

651 
I3O.2 
I3O.2 

92.5  CC. 
94.O 
76.O 
77.0 

92.5  mg. 
94.O 
QI.2 
92.4 

I.998 
I.967 
2.026 
2.000 

Mean,                     1.998 

It  appears,  therefore,  that  in  this  kind  of  solution  2  molecules  of  copper  oxide  are 
required  to  oxidize  1  molecule  of  creatinine.  The  2  molecules  of  copper  oxide 
yield  1  atom  of  oxygen.  It  will  be  recalled  that  5  molecules  of  copper  oxide  are 
used  up  in  oxidizing  1  molecule  of  dextrose.  For  equal  weights  the  reducing 
power  of  dextrose  is  about  twice  as  great  as  is  that  of  creatinine. 

Reducing  Power  of  Uric  Acid.  With  the  same  reagent  the  reducing  power 
of  uric  acid  may  be  measured,  the  uric  acid  being  dissolved  with  a  little  alkali  to 
form  a  soluble  urate.  The  table  below  illustrates  the  relation  of  the  reducing  and 
oxidizing  solutions : 


Uric  Acid  in 
100  cc. 

Copper 
Solution 
Taken. 

CuO  Equivalent. 

Uric  Acid 

Solution 

Used. 

Uric  Acid  to 
130.2  mg.  CuO. 

Mols  CuO 
to  1  Mol. 
C5H4N403. 

80  mg. 

80 

120 
120 

15.3  cc. 

25 
5° 
25 

39.8  mg. 
65.I 
130.2 
65.1 

36     CC. 

58 

76.8 

37-8 

94.2  mg. 
92.8 
9I.8 
90.8 

2.92 
2.96 
2.99 
3-°3 

Mean,  2.98 

From  this  it  appears  that  1  molecule  of  uric  acid  requires  3  molecules  of  copper 
oxide  for  oxidation  under  the  conditions  of  the  test,  or  1.5  atoms  of  oxygen. 
This  is  a  larger  amount  of  oxygen  than  would  be  required  for  the  oxidation  of  uric 
acid  to  urea  and  alloxan.     This  requires  1  atom : 

C5H4N403  +  O  +  H20  =  CON2H4  +  C4FLN204. 

But  secondary  reactions  also  take  place,  and  a  partial  oxidation  to  parabanic 
acid  may  be  represented  in  this  way : 

2C6H4N403  +  30  +  2H20  =  2CON2H4  +  QH2N204  +  C3H2N203  +  C02. 

This  possibly  represents  the  course  of  the  reaction  with  such  a  solution. 

With  these  reducing  values  established  it  is  possible  to  estimate  the  fraction  of 
the  total  urine  reduction  which  is  not  due  to  these  two  most  important  substances 
besides  sugar.  To  do  this  it  is  necessary  to  find  the  amount  of  uric  acid  and 
creatinine  in  a  given  volume  of  urine  and  determine  its  reducing  value  in  terms 
of  CuO  with  the  same  ammoniacal  solution.  If  the  reducing  power  of  the  crea- 
tinine and  uric  acid  be  subtracted  from  the  total,  the  reduction  due  to  glucose  and 
other  bodies  is  arrived  at.  To  illustrate,  in  the  examination  of  a  large  number  of 
urines  these  values  were  found : 


Total  reducing  power  of  1  cc.  of  urine  in  mg.  of  CuO  =  6.204 
Reducing  power  of  the  creatinine  present  in  same  terms  =  1.961 
Reducing  power  of  the  uric  acid  present  in  same  terms  =  0.935 
Reducing  power  of  the  sum  of  uric  acid  and  creatinine  =  2.896 


THE  ELECTRICAL  CONDUCTIVITY   OF   URINE.  309 

Nearly  one-half  of  the  total  reduction  then  corresponds  to  the  action  of  these 
two  nitrogen  bodies,  while  the  reduction  of  the  other  substances  is  equivalent  to 
3.308  mg.  of  CuO  per  cc.  If  this  is  calculated  as  glucose  it  represents  1.27  mg. 
per  cc.  As  several  other  bodies  contribute  to  this  reducing  action  the  value  of 
any  saccharine  substance  present  must  be  still  smaller.  It  has  been  found  by 
earlier  investigations  that  on  concentration  urine  loses  a  part  of  its  reducing  power. 
This  suggests  that  some  volatile  substances  may  be  responsible  for  part  of  it. 
It  should  be  mentioned  in  addition,  that  the  relative  extent  of  the  reducing  action 
varies  with  the  reagent  employed.  Knapp's  mercury  solution  has  been  used  for 
the  purpose  but  it  is  not  as  convenient  as  the  ammoniacal  copper  solutions. 

A  knowledge  of  this  normal  reducing  action  is  not  without  value, 
since  very  frequently  the  mistake  has  been  made  of  assuming  the  pres- 
ence of  sugar  in  suspicious  quantities  in  the  urine  merely  from  a  reduc- 
tion test.  In  some  of  the  extreme  cases  quoted  in  one  of  the  state- 
ments above,  the  normal  reduction  was  equivalent  to  over  0.3  per  cent 
of  glucose,  when  in  reality  it  was  largely  due  to  uric  acid  and  creati- 
nine. This  point  has  importance  in  clinical  observations.  In  another 
direction  also  the  question  is  important,  as  the  reducing  power  of  the 
urine  is  a  measure  of  the  extent  of  certain  kinds  of  excretion.  Crea- 
tinine and  uric  acid  are  probably  perfectly  normal  end  products  of 
metabolism,  and  when  large  in  amount  the  reduction  is  high.  It  will 
be  recalled  further  that,  as  mentioned  in  a  former  chapter,  strongly 
reducing  substances  are  produced  in  the  autolytic  changes  taking  place 
in  the  liver  and  pancreas.  In  cases  then,  this  "  normal "  reduction  may 
become  excessive. 

THE  ELECTRICAL  CONDUCTIVITY  OF  URINE. 

In  one  of  the  chapters  on  the  blood  the  nature  of  electrical  con- 
ductivity in  the  fluids  of  the  body  was  explained  and  the  methods  of 
determination  outlined.  As  this  conductivity  depends  mainly  on  the 
sum  of  the  inorganic  constituents  present,  and  as  sodium  chloride  is 
the  most  abundant  of  these,  the  determination  in  itself  has  but  a  lim- 
ited importance.  In  some  cases  the  value  of  the  conductivity  would  be 
merely  a  rough  measure  of  the  salt  consumed  with  the  food,  and  the 
salt  consumption  is  extremely  variable. 

Nearly  all  the  other  substances  found  in  the  urine  have  a  significance 
very  different  from  that  of  the  salt.  The  latter  is  consumed  and 
excreted  as  such,  while  the  other  important  urinary  constituents  are 
products  of  metabolism,  that  is,  of  the  breaking  down  of  the  digested 
and  absorbed  food  materials.  The  organic  products  of  metabolism 
are  practically  non-electrolytes  or  bodies  with  a  very  low  conducting 
power;  indeed  the  conductivity  of  a  weak  salt  solution  is  materially 
lowered  by  the  addition  of  urea  and  the  effect  of  the  purine  bodies  is 


3io 


PHYSIOLOGICAL    CHEMISTRY. 


practically  in  the  same  direction.  Aside  from  the  chlorides,  the  inor- 
ganic salts  of  the  urine  are  mainly  phosphates  and  sulphates  of  the 
alkali  or  alkali-earth  metals,  and  these  are  made  up  largely  from  the 
oxidation  of  sulphur  and  phosphorus  of  protein  foods.  We  consume 
a  certain  amount  of  phosphoric  and  sulphuric  acids  in  complex  organic 
combination,  in  the  lecithins  and  chondroitins,  for  example,  and  small 
amounts  of  mineral  sulphates  and  phosphates  are  also  found  in  some 
of  our  foods,  but  these  amounts  are  not  large  enough  to  vitiate  the 
truth  of  the  general  proposition  that  the  sulphuric  and  phosphoric  acids 
as  detected  in  the  urine  are  results  of  certain  kinds  of  metabolism. 
Now,  the  conductivity  measures  the  combined  effect  of  these  products 
of  oxidation  and,  if  it  could  be  determined  apart  from  the  effect  of  the 
chlorides,  a  factor  of  considerable  practical  importance  would  be 
secured. 

Approximately,  the  conductivity  due  to  metabolic  products  may  be 
found  by  subtracting  from  the  observed  conductivity  that  due  to 
sodium  chloride  in  the  same  solution.  The  chlorine  may  be  accurately 
determined  and  calculated  as  chloride;  then  from  tables  the  conduc- 
tivity for  a  solution  of  this  concentration  may  be  found  and  used  as  a 
correction  to  be  taken  from  the  total  observed  conductivity,  leaving 


B 

Period. 

A 

B 

A 

Volume 

Passed, 

cc. 

Specific 
Gravity 
200 

Conduc- 
tivity, K. 

Volume 

Passed, 

cc. 

Specific 
Gravity 
200 

Conduc- 
tivity, K. 

ervations   of  first 

y: 

retion  =  1,162  cc. 
uid     consumption 
1,750  cc. 

a 
.  0 
o'S 
0  a, 

»! 

II    C  0 

II  0  0 

E   U  m 

O            H 

si?  11 

6-  9  A.  M. 
9-12 
12-  3 
3-6 

6-  9  P.  M. 
9-6 

145 

178 

13s 
118 

1 28 
458 

1.024 

1.023 

1.024 
1.027 
1.026 

1.023 

O.02589 
O.O302I 
O.O2834 
O.O279O 
O.02694 
O.O242I 

112 
220 

77 

70 

198 

184 

I.022 
I.OI9 
I.026 
I.027 
1. 02 1 
I.026 

O.02264 
O.O2519 
0.02226 
0.02614 
O.02178 
0.02008 

-S  -S  S  .2*  11 

C     WiJ 

Means,  3-hr.  period 

1.024 

O.O2650 

1.0  4 

0.02228 

ervations  of   sec- 
d  day  : 

retion  =  1,265  re. 
uid     consumption 
2,100  cc. 

•   S 

H     0. 

B 

M     3 

II  § 

0    °   *" 

■£•0  "i 

85*11 

6-  9  A.  M. 
9-12 
12-  3 
3-6 

6-  9  P.  M. 
9-6 

192 
245 
155 
"3 
155 
405 

1.020 
1.020 
1.026 
1.026 

1.025 
I  025 

O.02645 
O.O2926 
O.02803 
O.02702 
O.O2702 
0.02I22 

410 
122 
96 
138 
1 80 
285 

I.OO7 
I. OI7 
I.O23 
I.O24 
1. 02I 
I.O24 

O.OIO39 
O.O2408 
O.02422 
O.O2355 
O.02144 
O.02369 

.a  0  x  .2"  II 

Means,  3-hr.  period 

1.024 

O.025I8 

I.020 

O.02184 

ervations  of  third 

y. 

.retion  —  1,324  cc. 
uid     consumption 
1,670  cc. 

g;S. 
0  £ 

m3 

II  gg 

'Z—  m 
<uI2  J" 
u   B   m 

a  1-11 

6-  9  A.  M. 
9-12 
12-  3 
3-6 

6-  9  P.M. 
9-6 

230 
260 
160 

134 
146 

394 

1.022 
1. 021 
1.026 
1.026 

1.025 
1.023 

O.O2683 
O.O2939 
O  02792 
O.O2755 
O.02580 
O.OI997 

278 

158 

92 

7i 
112 
388 

I-OI5 
I.022 
I  029 
I  O3O 
I.O27 
I. OI4 

O.O2629 
O.03037 
O.02720 
O.O2566 
O.02577 
O.OI492 

0   aj 

Means,  3-hr 

period 

1.024 

O.O2468 

I.O^I 

O.O225 1 

THE  ELECTRICAL  CONDUCTIVITY   OF  URINE. 


311 


the  desired  residual  or  metabolic  conductivity.  In  this  plan,  however, 
an  error  is  involved,  because  the  conductivity  of  the  chloride  taken 
from  the  tables  directly  is  that  found  in  pure  aqueous  solution  in 
absence  of  other  salts,  and  is  larger  than  the  true  conductivity  of  the 
chloride  as  it  exists  in  the  urine.  It  is  necessary,  therefore,  to  use  as 
a  correction  the  value  of  the  salt  conductivity,  not  in  aqueous  solution, 
but  in  a  solution  of  a  concentration  corresponding  to  that  of  urine. 

Several  attempts  have  been  made  to  measure  the  conductivity  of  mixtures  of 
electrolytes  and  formulate  the  results.  Most  of  the  experiments  have  been  made 
with  dilute  solutions  and  can  not  be  used  well  in  cases  like  the  present  one.  The 
author  has  investigated  the  question  with  special  reference  to  mixtures  like  the 
urine  and  has  considered  the  effect  of  urea  as  a  non-electrolyte  also.  The  con- 
ductivity varies  from  individual  to  individual,  and  from  hour  to  hour  according 
to  the  kind  and  amount  of  food  metabolized,  but  is  seldom  above  ^  =  0.03.  The 
table  (page  310)  illustrates  the  variations  in  the  urine  of  two  men,  both  well 
nourished  on  mixed  diet,  with  the  water  consumption  intentionally  high. 

By  making  complete  urine  analyses  and  duplicating  the  results  by  mixing  the 
inorganic  and  organic  ions  and  the  non-electrolytes  in  proper  proportion,  it  is  pos- 
sible to  reach  almost  exactly  the  corresponding  urine  conductivity  as  shown,  for 
example,  with  six  urines,  the  complete  analyses  of  which  were  employed  in  reach- 
ing the  mean  values  given  at  the  beginning  of  the  chapter. 


No.  of  Urine. 

1 

2                            3 

4 

5                           6 

k  as  observed. 
k  from  mixtures. 

0.02372 
0.02332 

O.02402 
O.024OO 

O.O2793 
O.02768 

O.OI984 
O.OI944 

O.O2898      I      O.O225I 
O.02886            O.O2158 

But  if  an  attempt  were  made  to  calculate  the  urine  conductivity  by  adding  together 
the  individual  conductivities  of  the  various  salts  for  the  concentrations  as  found 
by  analysis  the  result  would  be  too  high,  as  the  conductivity  of  each  substance 
is  lowered  somewhat  by  the  presence  of  the  others.  It  is  possible,  however,  by 
experimenting  with  known  artificial  mixtures,  to  determine  the  extent  of  this  modi- 
fication and  so  be  able  to  introduce  a  correction.  For  the  present  purpose  the 
disturbing  action  of  the  other  urinary  constituents  on  sodium  chloride  is  all  that 
is  called  for.  This  has  been  done,  but  the  details  of  the  investigation  can  not  be 
given  here. 

In  a  long  series  of  experiments  it  was  found  that  a  mean  correction  for  the 
effect  of  sodium  chloride  may  be  made  in  this  way.  The  chlorine  is  accurately 
determined  in  the  urine  and  calculated  as  sodium  chloride.  From  conductivity 
tables  the  value  of  «  for  the  calculated  concentrations  is  found  and  this  value  is 
diminished  by  3  per  cent  which  is  the  average  correction  due  to  the  presence  of 
other  substances,  organic  and  inorganic,  in  the  urine.  This  corrected  salt  con- 
ductivity in  turn  must  be  subtracted  from  the  observed  total  conductivity  to  find 
the  fraction  due  to  metabolic  products.  This  correction  gives  a  result  which  is  near 
the  truth  in  ordinary  normal  urines  and  which  may  be  taken  as  furnishing  a 
peculiar  kind  of  measure  of  the  total  metabolism,  that  will  be  found  to  have  a 
value  in  certain  calculations. 


312  PHYSIOLOGICAL    CHEMISTRY. 

THE  FREEZING  POINT  OF  URINE.     CRYOSCOPY. 

In  the  thirteenth  chapter  the  application  of  cryoscopic  methods  to 
blood  examinations  was  discussed,  and  the  apparatus  used  described. 
In  urine  investigations  also  freezing  point  determinations  have  become 
important  and  a  very  considerable  literature  has  accumulated.  The 
Beckmann  apparatus  may  be  employed,  as  with  the  blood,  but  the  Zikel 
modification  has  been  found  to  give  good  results  and  is  somewhat 
simpler  in  manipulation. 

Conductivity  and  cryoscopic  methods  do  not  yield  exactly  parallel  results ;  the 
conductivity  power  of  the  urine  depends  essentially  on  the  number  of  inorganic 
molecules  or  ions  present,  while  the  freezing  point  depression  depends  on  the  sum 
of  all  the  dissolved  substances.  Urea  is  therefore  important  in  the  one  instance, 
but  not  in  the  other,  as  it  is  practically  a  non-conductor.  This  being  the  case  it 
is  evident  that  valuable  information  may  be  obtained  by  a  combination  of  the  two 
methods,  as  it  is  possible  to  determine  the  fraction  of  the  osmotic  pressure  of 
the  urine  due  to  electrolytes  and  non-electrolytes.  Such  applications  are  fre- 
quently made. 

While  the  osmotic  pressure  of  the  blood  is  nearly  constant,  that  of  the  urine 
is  extremely  variable.  Ordinarily  the  limits  are  between  A  =  — 1-3°  and  — 2.00, 
but  after  great  water  consumption  on  the  one  hand,  or  consumption  of  much 
nitrogenous  food,  or  salt,  without  sufficient  liquid,  on  the  other,  the  freezing  point 
of  the  urine  may  vary  from  A  =  —  o.r°  to  — 3.00.  That  is,  the  urine  concen- 
tration may  range  from  one-fifth  that  of  the  blood  to  over  five  times  the  blood 
concentration,  expressed  in  active  molecules.  It  will  be  remembered  that  a  freez- 
ing point  depression  of  i°  C.  corresponds  to  an  osmotic  pressure  of  12.1  atmo- 
spheres. 

The  applications  of  this  cryoscopic  method  to  urine  are  mainly  in  the  direction 
of  diagnosis.  Since  it  is  possible  to  collect  the  urine  from  each  kidney  separately, 
by  ureter  catheterization  or  equivalent  means,  a  test  of  the  two  portions,  will  dis- 
close any  difference  in  the  performance  of  the  two  organs.  Normally,  the  secre- 
tions from  the  two  kidneys,  for  a  given  time,  should  be  the  same.  A  freezing 
point  determination  is  easily  made  and  will  show  if  one  kidney  is  doing  more  work 
than  the  other.  By  including  a  conductivity  test  it  may  be  found  that  the  diffi- 
culty in  excretion  is  more  pronounced  for  one  class  of  substances  than  for 
another. 


CHAPTER   XX. 

SOME  PRACTICAL  URINE  TESTS. 

In  this  chapter  brief  directions  will  be  given  for  the  routine  exami- 
nation of  urine,  such  as  is  called  for  in  clinical  observations.  In  such 
work  the  color  and  reaction,  already  referred  to,  are  always  noted,  and 
the  specific  gravity. 

SPECIFIC  GRAVITY. 

For  very  accurate  work  the  specific  gravity  of  the  urine  is  determined  by  means 
of  the  pycnometer  or  weighing  bottle,  but  in  the  ordinary  clinical  practice  it  is 
customary  to  employ  the  urinometer,  which  may  furnish  very  accurate  results  if 
the  instrument  is  properly  made  and  graduated.  Formerly  the  reading  was  based 
on  a  temperature  of  about  15. 5°  in  the  urine,  referred  to  water  of  the  same  tem- 
perature as  unity.  A  much  more  rational  plan  is  to  assume  a  standard  tempera- 
ture of  25°  C.  in  the  urine  and  refer  the  reading  to  water  at  40  C.  as  the  unit. 

From  a  test  of  the  specific  gravity  it  is  possible  to  make  an  approximate  estimate 
of  the  total  solids  present.  From  a  large  number  of  observations  made  in  the 
author's  laboratory  it  has  been  found  that  by  multiplying  the  last  two  figures  of 
the  specific  gravity  at  250  C,  by  2.6  a  product  is  secured  which  represents  the 
weight  of  urinary  solids  in  1000  cc.  This  2.6  is  Long's  coefficient,  and  takes  the 
place  of  the  Haeser  coefficient  based  on  tests  at  a  lower  temperature.  Thus,  if 
the  specific  gravity  at  25°  C.  is  1.023  (Sp.  gr.  -"  =  1.023)  we  have  23X2.6  =  59.8, 
or  the  number  of  grams  of  solids  per  liter.  The  direct  determination  of  total 
solids  by  evaporation  and  weighing  is  not  perfectly  accurate,  because  of  losses 
through  partial  decomposition  of  some  of  the  constituents. 

The  specific  gravity  of  the  urine  varies,  naturally,  with  the  food  consumption 
and  volume  of  liquid  consumed.  It  may,  normally,  be  as  low  as  1.010  and  as  high 
as  1.030  or  perhaps  higher.  A  diet  rich  in  meats  gives  rise  to  a  urine  with  high 
specific  gravity  because  of  the  resultant  urea  excretion ;  much  salt  in  the  food  has 
the  same  effect.  On  the  other  hand,  a  diet  rich  in  fats  and  carbohydrates,  with 
low  proteins,  gives  rise  to  a  urine  with  low  specific  gravity,  because  in  such  cases 
little  is  formed  to  be  excreted  by  the  kidneys.  With  ordinary  mixed  diet  a  spe- 
cific gravity  of  1.020  to  1.025  is  generally  observed. 

In  diabetes  mellitus  the  specific  gravity  of  the  urine  may  be  very  high,  from  the 
excretion  of  sugar,  as  will  be  pointed  out  below.  But  it  must  be  kept  in  mind 
that  a  specific  gravity  high  enough  to  suggest  sugar  may  be  reached  through  high 
salt  and  protein  and  low  water  consumption. 

The  clinician  is  not  so  much  interested  in  the  normal  constituents  of 
the  urine  as  he  is  in  the  possible  presence  of  bodies  pointing  to  a  patho- 
logical condition  in  his  patient.  A  marked  variation  in  some  of  the 
normal  constituents  of  the  urine  may  also  have  an  important  bearing 
on  the  diagnosis,  and  he  must  know  how  to  make  these  tests.  Follow- 
ing the  usual  routine,  we  shall  take  up  albumin  and  sugar  first. 

3'3 


314  PHYSIOLOGICAL    CHEMISTRY. 

THE  TESTS  FOR  ALBUMINS. 

Albumins  are  present  in  normal  urine  in  traces  only,  and  such  traces  are  not 
detected  by  the  usual  reagents  described  below.  Albumin  in  the  urine  in  any 
appreciable  amount  is  indicative  of  a  pathological  condition,  and  may  be  due  to  a 
number  of  causes.  A  great  number  of  tests  have  been  suggested  for  the  recogni- 
tion of  this  pathological  albumin,  and  these  all  depend,  practically,  on  the  fact 
that  the  soluble,  invisible  protein  may  be  easily  coagulated  in  the  urine  and  so 
rendered  visible.     The  ordinary  serum  albumin  will  be  considered  first. 

Heat  Test.  At  a  temperature  near  70°  C.  the  serum  albumin  becomes  coagulated 
and  opaque.  Heat  a  few  cc.  of  the  urine  in  a  test-tube  nearly  to  the  boiling  point. 
If  the  liquid  becomes  cloudy  albumin  is  suggested.  But  the  urine  usually  contains 
enough  so-called  earthy  phosphates  to  become  cloudy  on  heating,  and  this  cloud 
resembles  the  albumin  coagulation  very  closely.  To  avoid  a  mistake  here  the 
urine  must  be  very  weakly  acidified  with  dilute  acetic  acid  before  warming.  This 
prevents  the  precipitation  of  phosphates  but  does  not  interfere  with  the  albumin 
coagulation.     Avoid  using  much  acid. 

Nitric  Acid  Test.  In  contact  with  strong  nitric  acid  the  albumin  of  the  urine 
is  immediately  coagulated.  To  make  the  test  pour  two  or  three  cc.  of  strong  nitric 
acid  into  a  narrow  test-tube,  warm  slightly  and  by  means  of  a  dropping-tube 
introduce  over  the  acid  a  layer  of  the  clear  urine  (filtered  previously,  if  not 
clear).  In  presence  of  albumin  a  cloudiness,  or  even  a  heavy  precipitate,  appears 
at  the  junction  of  the  two  liquids.  This  is  an  extremely  delicate  test  and  is  very 
commonly  employed  clinically.  The  nitric  acid  is  warmed  to  prevent  the  possible 
precipitation  of  urea  nitrate,  which  occasionally  happens  with  cold,  very  con- 
centrated urine.  A  simple  change  of  color  at  the  junction  layer  is  not  due  to 
albumin.     The  latter  yields  an  actual  coagulation. 

Picric  Acid  Test.  Many  reagents  besides  nitric  acid  have  the  power  of  forming 
precipitates  with  albumin.  A  saturated  aqueous  solution  of  picric  acid  is  very 
useful  for  this  purpose,  and  when  added  to  albuminous  urine  produces  a  yellowish 
white  cloud  or  precipitate.  It  is  best  to  acidify  the  urine  slightly  with  acetic 
acid  first.  If  this  causes  a  precipitate,  of  mucin  possibly,  filter  it  off  and  then 
add  the  picric  acid,  drop  by  drop.  The  test  is  extremely  delicate,  but  is  not 
better  than  the  preceding  one. 

Double  Iodide  Test.  This  is  known  also  as  Tanret's  Test  and  the  Mercuro- 
Potassium  Iodide  Test.  The  reagent  used  is  made  by  dissolving  33.1  gm.  of 
potassium  iodide  in  distilled  water  and  adding  to  it  gradually  13.5  gm.  of  mer- 
curic chloride.  The  mixture  is  stirred  until  all  is  dissolved,  and  diluted  to  make 
800  cc.  To  this  100  cc.  of  pure,  strong  acetic  acid  is  added.  If  a  slight  pre- 
cipitate remains  decant  carefully  and  dilute  to  one  liter  with  distilled  water.  The 
solution  contains  approximately  4  KI  to  HgCl2. 

To  make  the  test  filter  the  urine,  if  not  clear,  and  add  enough  acetic  acid  to 
give  a  sharp  reaction.  If  a  precipitate  or  turbidity  now  appears,  possibly  due 
to  mucin  bodies,  filter  again  and  to  10  cc.  of  the  clear  filtrate  add  a  few  drops 
of  the  reagent.  In  presence  of  proteins  a  white  cloud  is  formed,  or  even  a  pre- 
cipitate if  much  of  the  albumin  substance  is  in  the  solution. 

The  test  is  extremely  delicate,  but  it  must  be  remembered  that  the  same  reagent 
is  employed  in  testing  for  alkaloids.  If  the  urine  happens  to  contain  traces  of 
those  bodies,  from  previous  medication,  a  reaction  will  certainly  follow.  But 
such  precipitates  may  be  distinguished  by  their  solubility  in  alcohol.  A  precipitate 
may  be  formed  also  in  presence  of  excess  of  urates.  Such  a  precipitate  is  dissi- 
pated by  heating ;  it  may  be  avoided  in  the  first  place  by  proper  dilution  of  the  urine. 

The  Amount  of  Albumin.  It  is  not  alone  sufficient  that  we  are  able  to  detect 
the  presence  of  albumin  in  urine;  we  often  need  to  know  its  amount  to  determine 


SOME    PRACTICAL    URINE    TESTS.  3  I  5 

the  practical  value  of  a  line  of  treatment  pursued  from  day  to  day.  To  be  of 
the  greatest  possible  service,  a  method  must  be  so  easy  of  execution  that  approxi- 
mately correct  results  may  be  obtained  by  it  by  the  use  of  simple  apparatus  and  in 
a  short  time.  Several  methods  are  known  by  which  the  amount  of  albumin  in 
urine  can  be  found.  One  of  these,  and  the  best,  may  be  called  the  gravimetric 
method,  as  by  it  the  albumin  is  precipitated,  collected,  and  weighed.  In  another, 
the  albumin  is  precipitated  and  its  volume  measured,  while  in  a  third  process  the 
amount  of  albumin  is  estimated  from  the  degree  of  turbidity  caused  by  its  pre- 
cipitation  in  the  urine. 

The  gravimetric  method  consists  essentially  in  coagulating  the  albumin,  collect- 
ing it  on  some  form  of  filter,  and,  after  proper  washing,  weighing  the  pre- 
cipitate. This  method  may  be  made  to  give  very  good  results  but  is  too  difficult 
and  tedious  for  the  clinical  laboratory. 

Volume  Methods.  One  of  the  simplest  of  these  is  the  one  proposed  by  Esbach. 
In  this  a  special  tube  is  used,  called  the  Esbach  albuminometer,  and  a  special  solu- 
tion or  reagent  made  by  dissolving  10  grams  of  pure  picric  acid  and  20  grams  of 
pure  citric  acid  in  a  liter  of  distilled  water.  The  solution  must  be  filtered  if  it  is 
not  perfectly  clear,  and  is  the  same  as  the  one  used  for  the  qualitative  test.  The 
principle  involved  in  the  employment  of  the  method  is  this :  The  precipitate  of 
albumin  and  picric  acid  settles  in  coherent  manner  and  in  a  compact  volume  pro- 
portional to  its  weight,  provided  certain  definite  amounts  of  the  reagent  and  urine 
are  taken.  The  albuminometer,  or  measuring  tube  used,  resembles  a  test-tube  of 
heavy  glass  about  six  inches  long  and  is  graduated,  empirically,  to  show  how  much 
urine  and  reagent  to  take  and  the  amount  of  albumin  obtained  in  grams  per  liter, 
or  tenths  of  1  per  cent. 

The  test  is  carried  out  in  this  manner: 

Urine  is  poured  in,  to  the  mark,  and  then  the  reagent,  described  above,  to  its 
proper  level.  The  tube  is  closed  with  the  thumb  and  tipped  backward  and  for- 
ward eight  or  ten  times  until  the  liquids  are  thoroughly  mixed.  It  is  then  closed 
with  a  rubber  stopper  and  allowed  to  stand  in  a  perpendicular  position  twenty- 
four  hours.  This  will  give  the  precipitate  time  to  settle  thoroughly  after  which 
the  amount  can  be  read  off  on  the  scale.  The  results  are  accurate  enough  for 
clinical  purposes  and  by  practice  can  be  made  to  agree  moderately  well  with  those 
found  by  the  gravimetric  method.  But  the  tube  must  not  be  violently  shaken,  and, 
as  the  test  is  an  empirical  one,  it  should  not  be  allowed  to  stand  much  longer  than 
one  day  before  reading  the  result. 

In  any  case  in  applying  this  test  the  urine  should  not  be  highly  concentrated. 
The  best  results  are  obtained  with  urine  of  low  specific  gravity,  and  with  the 
albumin  not  over  0.3  per  cent.  When  the  test  shows  an  amount  in  excess  of  this, 
the  urine  should  be  diluted  and  a  new  trial  made. 

A  yellowish-red  precipitate  which  sometimes  separates  on  long  standing  must 
not  confuse  the  analyst.     It  consists  of  uric  acid. 

The  Tests  for  Globulins.  These  protein  bodies  resemble  the  albumins  in  many 
respects,  and  like  them  occur  only  pathologically  in  the  urine.  In  all  the  pre- 
ceding tests,  globulins  react  as  do  the  albumins.  Among  the  distinctive  tests  one 
only  need  be  mentioned  here. 

Dilution  Test.  Globulin  is  insoluble  in  water,  but  soluble  in  dilute  salt  solutions; 
— hence  its  solubility  in  urine.  If  the  latter  is  diluted  until  the  specific  gravity  is 
1.002  or  1.003  the  globulin  may  separate  out.  At  any  rate  the  addition  of  a  few 
drops  of  dilute  acetic  acid  will  produce  the  desired  result.  A  current  of  carbon 
dioxide  passed  into  the  diluted  liquid  for  several  hours  accomplishes  the  same 
end. 

The  test  may  be  modified  in  this  manner.     Filter  the  urine  if  it  is  not  perfectly 


316  PHYSIOLOGICAL    CHEMISTRY. 

clear,  and  then  pour  it,  drop  by  drop,  into  a  tall,  narrow  beaker  of  distilled  water. 
If  globulin  is  present  it  is  thrown  out  as  a  white  cloud  which  shows  as  the  drops 
pass  down  through  and  mix  with  the  lighter,  clear  water.  The  globulin  may 
afterward  be  confirmed  by  adding  a  small  amount  of  salt  solution  which  will  cause 
the  precipitate  to  disappear. 

PEPTONES  AND  PROTEOSES. 

True  peptones  are  very  rarely  found  in  urine.  The  bodies  described  as  peptones 
are  probably  highly  converted  proteoses,  or  albumoses  of  the  deuteroalbumose  type. 

The  recognition  of  albumose  is  not  a  matter  of  difficulty,  as  it  is  distinguished 
from  the  other  protein  compounds  sometimes  found  in  the  urine  by  several  well- 
marked  characteristics.  It  is  not  coagulated  by  heat  or  by  the  addition  of  acetic 
or  warm  nitric  acid,  and  is  very  soluble  in  hot  water.  It  is  much  less  soluble  in 
cold  water,  but  the  presence  of  small  amounts  of  salts  seems  to  increase  its  solu- 
bility here  in  marked  degree. 

In  presence  of  albumin  or  globulin  it  can  be  found  by  the  following  process 
unless  it  is  in  very  small  amount.  The  urine  is  saturated  with  pure  sodium 
chloride,  which  will  precipitate  albumose  if  present,  and  then  enough  acetic  acid 
is  added  to  give  a  very  strong  acid  reaction.  The  mixture  is  boiled  and  filtered 
hot.  This  treatment  throws  out  both  albumin  and  globulin,  and  redissolves  a  pre- 
cipitate of  albumose  which  may  have  formed.  The  latter  would  therefore  be  found 
in  the  clear  filtrate  and  sometimes  in  amount  sufficient  to  precipitate  as  this  cools. 
The  filtrate  should  therefore  be  allowed  to  remain  at  rest  until  quite  cool.  If  much 
albumose  is  present  it  will  appear  as  a  white  cloud.  Sometimes,  however,  it  will 
be  necessary  to  concentrate  the  filtrate  before  looking  for  this  reaction,  and  this 
is  done  by  evaporating  slowly  on  the  water-bath  to  half  the  volume.  On  now 
cooling,  salt  will  quickly  settle  out  while  the  albumose  precipitates  later  in  floc- 
culent  form.  After  the  salt  has  separated  the  liquid  may  be  tested  by  the  biuret 
reaction,  described  in  earlier  chapters. 

Another  test  is  this :  Separate  the  albumin  and  globulin  by  boiling  with  a  small 
amount  of  acetic  acid  without  the  salt.  Filter  while  warm  and  concentrate  the 
filtrate  to  a  volume  of  one-third.  Allow  to  cool  thoroughly  and  add  a  large  excess 
of  saturated  solution  of  ammonium  sulphate.  This  gives  a  white  flocculent  pre- 
cipitate of  albumose,  if  present.  The  precipitate  can  be  collected  on  a  filter  and 
washed  with  the  saturated  ammonium  sulphate  solution  and  then  dissolved  in  a 
little  distilled  water,  poured  on  the  filter.  This  filtrate  gives  tests  with  picric  acid, 
potassium  ferrocyanide  and  acetic  acid,  and  other  albumin  reagents. 

If  the  original  urine  shows  no  reactions  for  albumin  or  globulin  the  albumose 
tests  can  be  applied  directly  after  concentration.  The  method  by  precipitation  by 
means  of  picric  acid  gives  good  results.  The  biuret  test  is  also  delicate  if  the 
urine  is  clear  and  of  light  color. 

THE  TESTS  FOR  SO-CALLED  MUCIN. 

Much  confusion  exists  at  the  present  time  as  to  the  exact  nature' of  the  protein 
bodies  in  the  urine  which  have  long  been  known  as  mucins.  The  true  mucins 
belong  to  the  group  of  gluco-proteids  and  are  seldom  found  in  the  urine,  but 
certain  other  protein  bodies  are  practically  always  present  normally,  and  with 
them  several  acid  substances  which,  under  the  proper  conditions,  are  able  to 
precipitate  these  normal  traces  of  protein.  Among  these  acids  chondroitin-sulphuric 
acid  is  the  most  important,  and  in  acetic  acid  solution  it  combines  with  and  pre- 
cipitates the  normal  protein.  Sometimes  the  trace  of  protein  normally  present  is 
so    small    that    no    precipitate    forms    on    slightly    acidifying.     In    such    cases    the 


SOME    PRACTICAL    URINE    TESTS.  3 17 

addition  of  a  little  albumin  solution  to  the  acidified  urine  usually  produces  a  pre- 
cipitate, as  the  albumin-precipitating  agent  is  ordinarily  present  in  sufficient  amount. 

The  kind  of  albumin  which  may  be  present  and  give  this  reaction  is  not  clearly 
defined.  In  some  cases  it  appears  to  be  a  nucleo-albumin,  and  occasionally  a  gluco- 
proteid,  which  yields  a  reducing  substance  on  boiling  with  acids.  In  working  with 
a  large  volume  of  urine  it  is  sometimes  possible  to  separate  from  the  so-called 
mucin  precipitate  a  reducing  substance  suggesting  the  gluco-proteid;  in  other 
cases  this  precipitate  may  be  free  from  sulphate  or  reducing  substance,  but  contain 
a  phosphate-yielding  complex  which  can  point  to  a  nucleo-albumin  or  nucleo-proteid. 
These  reactions  are  too  complex  for  ordinary  clinical  demonstration. 

Acetic  Acid  Test.  The  addition  of  an  excess  of  acetic  acid,  that  is  enough 
to  give  a  strong  acid  reaction  and  make  up  about  0.2  per  cent,  of  the  whole  volume, 
produces  a  flocculent  precipitate  in  presence  of  the  "  mucin  "  substance.  Heat  is 
not  applied  in  this  test,  and  its  delicacy  may  be  increased  by  dilution  of  the  urine. 

Citric  Acid  Test.  In  this  test  a  layer  of  the  urine  is  poured  over  a  concentrated 
solution  of  citric  acid.  The  "mucin"  appears  as  a  cloud  at  the  junction  of  the 
two  liquids. 

In  pouring  urine  over  nitric  acid  a  mucin  cloud  or  band  sometimes  makes  its 
appearance  about  a  centimeter  above  the  junction  point.  In  this  test  albumin 
shows  as  a  cloud  at  the  junction  point.  In  these  acid  tests  the  precipitation  of 
uric  acid  is  prevented  by  the  dilution  with  two  or  three  volumes  of  water. 

THE  TESTS  FOR  SUGARS. 

Normal  urine  contains  a  trace  of  carbohydrate,  probably  glucose,  which  may 
be  recognized  by  very  delicate  tests.  Pathologically  glucose,  and  sometimes  other 
sugars,  occur  in  quantity  and  may  be  identified  by  a  great  number  of  tests.  We 
have  to  distinguish  then,  between  what  is  known  as  a  physiological  glycosuria, 
and  a  pathological  condition  most  commonly  characteristic  of  diabetes  mellitus. 
In  the  normal  urine  the  trace  of  sugar  may  not  be  over  one-twentieth  of  one  per 
cent.,  while  in  advanced  diabetes  it  may  amount  to  five  per  cent.,  or  even  more, 
with  a  volume  of  five  or  six  liters  daily. 

The  most  convenient  of  our  sugar  tests  are  based  on  the  fact  that  the  usually- 
occurring  glucose  is  an  aldehyde  body  and  responds  to  certain  characteristic  tests. 
Fructose,  sometimes  present,  is  a  ketone  sugar,  and  responds  to  the  same  tests  in 
general.  Among  these  we  have,  first,  the  so-called  reduction  tests,  the  best  of 
which  are  as  follows : 

Moore's  Test.  This  depends  on  the  reaction  between  reducing  sugars  and  strong 
alkali  solutions.  When  a  solution  of  sugar  or  diabetic  urine  is  mixed,  without 
heating,  with  a  solution  of  sodium  or  potassium  hydroxide,  no  change  is  at  first 
apparent  unless  the  amount  of  sugar  present  is  large  or  the  alkali  very  strong. 
But  on  application  of  heat,  even  with  weak  sugar  solutions,  a  yellow  color  soon 
appears  which  grows  darker,  becoming  yellowish  brown,  brown,  and  finally  almost 
black,  while  an  odor  of  caramel  is  quite  apparent.  The  strong  alkali-sugar  solu- 
tion absorbs  atmospheric  oxygen,  giving  rise  to  a  number  of  products  among  which 
lactic  acid,  formic  acid,  pyrocatechol  and  others  have  been  recognized.  The  brown 
color  is  due  to  other  unknown  decomposition  products. 

This  is  a  good  reaction  for  all  but  traces  of  sugar,  as  the  intense  dark  brown 
color  and  strong  odor  are  not  given  by  other  substances  liable  to  be  present  in 
urine. 

But  traces  of  sugar  cannot  be  recognized  by  this  test  with  certainty,  as  the 
color  of  normal  urine  even  is  darkened  to  some  extent  by  the  action  of  alkalies. 

Urine  containing  much  mucin  becomes  perceptibly  darker  when  heated  with 
sodium,  potassium,  or  calcium  hydroxide  solutions. 


3.1  8  PHYSIOLOGICAL    CHEMISTRY. 

The  Trommer  Test.  This  is  one  of  the  oldest  and  best  known  of  the  tests 
for  the  recognition  of  sugar  in  urine,  and  is  a  typical  reduction  test.  It  is  per- 
formed by  adding  to  the  urine  an  equal  volume  of  10  per  cent,  solution  of  sodium 
or  potassium  hydroxide  and  then  a  very  few  drops  (three  or  four  to  begin  with) 
of  dilute  solution  of  copper  sulphate  as   described  in  Chapter  III. 

The  test  must  be  used  with  certain  precautions.  If  albumin  is  present  it  must 
be  coagulated  and  filtered  out.  The  amount  of  copper  sulphate  used  must  be  small, 
because  if  only  a  trace  of  sugar  is  present  and  much  copper  is  used  the  latter  will 
give  a  blue  precipitate  which  can  not  redissolve,  and  which  turns  black  on  boiling, 
thus  obscuring  a  sugar  reaction  which  may  be  given  at  the  same  time.  In  mak- 
ing this  test  if  a  black  precipitate  is  thus  found  it  must  be  repeated,  using  less  of 
the  copper. 

The  active  body  in  producing  the  reaction  is  copper  hydroxide,  but  this  must 
be  in  solution  to  act  as  a  good  oxidizing  agent  with  sugar;  and  the  test,  therefore, 
becomes  uncertain  or  unsatisfactory  if  so  much  copper  is  added  that  the  hydroxide 
formed  cannot  be  dissolved  by  the  sugar  which  may  be  present.  In  doubtful 
cases  it  becomes  necessary  to  make  several  trials  before  the  right  proportion  between 
urine,  alkali,  and  copper  solution  is  found.  In  the  solution,  on  completion  of  the 
reaction,  several  oxidation  products  of  sugar  are  found,  among  which  are  formic 
acid,  oxalic  acid,  tartronic  acid,  etc.  But  the  complete  reaction  is  obscure.  In 
order  to  avoid  the  indicated  uncertainty  of  the  Trommer  test  when  used  for  small 
amounts  of  sugar  the  next  one  was  proposed. 

The  Fehling  Solution  Test.  Fehling  suggested  the  use  of  a  solution  containing 
along  with  the  copper  sulphate  and  alkali  a  tartrate  to  dissolve  the  copper  hydroxide 
formed  by  the  first  two.  Many  substances  besides  sugars  have  the  power  of  dis- 
solving copper  hydroxide  with  a  deep  blue  color.  Among  these  may  be  mentioned 
tartaric  acid  and  the  tartrates,  glycerol,  mannitol  and  others  of  less  value. 

A  solution  prepared  by  mixing  certain  quantities  of  alkali,  copper  sulphate,  and 
either  one  of  these  bodies,  with  water  in  definite  proportions,  remains  perfectly 
clear  when  boiled.  But  if  a  trace  of  glucose  (or  several  other  sugars)  is  present 
the  usual  yellow  precipitate  forms. 

In  performing  the  test  with  the  Fehling  solution  described  in  Chapter  III  three 
or  four  cubic  centimeters  of  the  solution  are  poured  into  a  test-tube,  diluted  with 
an  equal  volume  of  water,  and  boiled.  The  solution  must  remain  clear.  Then  the 
urine  is  poured  in,  a  few  drops  at  a  time,  and  the  mixture  is  boiled.  If  sugar  is 
present  in  the  urine  in  an  amount  above  one-fifth  of  one  per  cent,  it  should  show 
at  once  with  this  treatment.  If  but  a  trace  of  sugar  is  present,  more  urine  must 
be  added  and  the  boiling  repeated. 

When  normal  urine  is  heated  with  Fehling  solution  a  greenish  flocculent  pre- 
cipitate usually  makes  its  appearance.  This  has  no  significance,  as  it  is  due  to  the 
phosphates  normally  present,  which  come  down  when  the  reaction  is  made  alka- 
line. Many  urines  produce  a  clear  dark  green  solution  when  heated  with  the  Feh- 
ling solution.  This  is  a  partial  reduction  reaction  and  like  the  other  has  no  spe- 
cial importance,  as  urines  free,  from  sugar  give  it.  At  other  times  urines  free 
from  sugar  yield  an  almost  colorless  mixture  when  boiled  with  the  Fehling  solu- 
tion. These  peculiar  reduction  effects  are  due  to  the  presence  of  uric  acid,  crea- 
tinine, pyrocatechol  and  several  other  substances  and  are  generally  characterized  by 
discharge  of  the  deep  blue  color  of  the  solution  without  precipitation  of  the  copper 
suboxide.  Certain  substances  taken  as  remedies  give  rise  to  products  in  the  urine 
which  exert  a  similar  action.  Occasionally,  however,  the  amount  of  uric  acid  is  so 
large  that  the  reduction  is  accompanied  by  actual  precipitation  of  the  copper  as 
red  oxide.  This  fact  is  of  interest  as  it  makes  the  test,  at  times,  somewhat  uncertain, 
but  it  is  a  very  simple  matter  to  determine  whether  or  not  a  great  excess  of  uric 


SOME    PRACTICAL    URINE    TESTS.  3  1 9 

acid  is  present,  as  will  be  pointed  out  later.  The  liability  to  error  in  the  Trommer 
test  from  these  causes  is  less  than  in  the  Fehling  test,  but  notwithstanding  this 
the  latter  must  still  be  regarded  as  the  better  test  practically,  because  of  its  great 
convenience  and  the  sharpness  of  the  reaction  with  even  traces  of  sugar.  The 
ingredients  of  the  Fehling  test  are  best  kept  in  separate  bottles  closed  with  rubber 
stoppers,  as  the  mixed  solution  does  not  keep  well,  as  ordinarily  prepared.  It  is 
customary  to  keep  the  copper  sulphate  in  one  bottle  and  the  alkali  and  tartrate  in 
another. 

Several  other  copper  test  solutions  are  in  use;  the  Loewe  solution  contains 
glycerol  in  place  of  sodium-potassium  tartrate  of  the  Fehling  solution,  while  man- 
nitol  is  employed  for  the  same  purpose  in  the  Schmiedeberg  solution.  The  reduc- 
tion is  the  same  as  in  the  Fehling  test. 

The  Bismuth  Test.  Bottger  found  (1856)  that  in  the  presence  of  alkali,  bis- 
muth subnitrate  is  reduced  to  the  metallic  condition  by  the  action  of  glucose  in 
hot  solution.  As  a  urine  test  he  recommended  to  make  it  strongly  alkaline  with 
sodium  carbonate,  and  then  add  a  very  small  amount,  what  can  be  held  on  the 
point  of  a  penknife,  of  the  pure  bismuth  subnitrate.  On  boiling  the  mixture  the 
insoluble  bismuth  compound,  which  settles  to  the  bottom,  turns  dark  if  sugar  is 
present. 

The  test  is  at  present  carried  out  by  adding  to  the  urine  in  a  test-tube  an  equal 
volume  of  10  per  cent,  solution  of  sodium  or  potassium  hydroxide,  and  then  the 
subnitrate.  Boiling  gives  the  reaction  as  before.  In  absence  of  sugar  (or  albu- 
min) the  bismuth  compound  remains  white. 

In  performing  this  test  only  a  very  small  amount  of  the  subnitrate  should  be 
taken.  This  is  absolutely  necessary  in  the  detection  of  traces  of  sugar.  In  this 
case  the  reduction  is  but  slight,  and  not  much  black  powder  of  bismuth  or  its 
oxide  can  be  formed.  If  a  great  excess  of  the  white  subnitrate  is  taken  it  may 
be  sufficient  to  completely  obscure  the  reduction  product.  It  is  frequently  well  to 
use  not  more  than  four  or  five  milligrams  of  the  subnitrate. 

The  black  precipitate  formed  was  at  one  time  supposed  to  be  finely  divided 
metallic  bismuth.  Later  investigations  seem  to  show  that  it  consists  essentially 
of  lower  oxides  of  bismuth.  This  test  has  certain  advantages  over  the  copper 
tests.  It  is  easily  made,  and  with  materials  everywhere  obtainable  in  condition  of 
sufficient  purity.  Furthermore,  the  reaction  is  not  given  with  uric  acid,  which  it 
will  be  remembered  may  act  on  the  Fehling  solution  if  excessive. 

Albumin,  however,  interferes  with  the  test,  as  it  gives,  also,  a  black  precipitate 
when  boiled  with  alkali  and  the  bismuth  subnitrate.  In  this  case  the  albumin 
gives  up  sulphur  and  forms  bismuth  sulphide ;  if  it  is  present  in  a  urine  it  should 
be  coagulated  and  filtered  out  before  trying  the  test. 

Fallacies  in  the  Reduction  Tests.  It  has  been  shown  above  that  several  bodies 
normally  found  in  urine  are  able  to  reduce  the  alkaline  copper  solutions.  Some 
of  these  interfere  with  the  bismuth  reactions  also,  but  not  to  the  same  degree;  but 
attention  must  be  called  to  another  source  of  error  which  is  very  important.  In 
warm  weather  it  is  often  desirable  to  add  something  to  urine  to  prevent  its  rapid 
decomposition,  and  several  substances  have  been  suggested  for  the  purpose.  The 
best  known  are  chloral,  chloroform,  salicylic  acid,  phenol,  and  formaldehyde.  Un- 
fortunately all  of  these  except  phenol  have  a  rather  marked  action  on  the  copper 
solutions.  As  a  preservative  phenol  is  objectionable  from  other  standpoints.  Urine 
intended  for  sugar  tests  should  be  tested  as  soon  as  possible  after  collection,  and 
no  foreign  substances  should  be  added  as  a  preservative.  Neglect  of  this  very 
simple  and  obvious  precaution  has  caused  many  serious  blunders,  especially  in  the 
examination  of  urine  from  applicants  for  life  insurance. 

The  Phenylhydrazine  Test. — In  this  test  a  reaction  discovered  some  years  ago 


320  PHYSIOLOGICAL    CHEMISTRY. 

has  been  applied  by  v.  Jaksch  to  the  examination  of  urine.  Add  to  about  10  cc. 
of  urine  0.2  gram  of  phenylhydrazine  hydrochloride  and  a  slightly  greater  amount 
of  sodium  acetate.  Warm  the  mixture  gently,  and  if  solution  does  not  take  place 
add  half  the  volume  of  water  and  heat  half  an  hour  on  the  water-bath.  Then  cool 
the  test-tube  by  placing  it  in  cold  water  and  allow  it  to  stand.  If  sugar  is  present 
a  yellow  precipitate  settles  out,  which  consists  of  minute  needles  generally  arranged 
in  rosettes,  visible  under  the  microscope.  Albumin  does  not  obscure  this  test,  but 
if  much  is  present  it  is  best  to  coagulate  it  as  well  as  possible  by  heating,  and  then 
filter.  The  yellow  precipitate  is  phenyl-glucosozone.  The  reaction  is  not  much  used 
in  urine  analysis,  but  is  of  great  value  in  the  identification  of  sugar  under  other 
conditions. 

The  Fermentation  Test. — When  yeast  is  added  to  urine  containing  sugar  and 
the  mixture  left  in  a  moderately  warm  place  the  usual  fermentation  soon  begins 
which  is  shown  by  two  principal  changes.  Carbon  dioxide  is  given  off,  which  may 
be  collected  and  identified,  and  the  mixture  becomes  lighter  in  specific  gravity. 
When  only  traces  of  sugar  are  present  the  test  by  collection  and  identification  of 
the  carbon  dioxide  frequently  fails  because  of  the  solubility  of  the  gas  in  the  liquid. 

The  variation  in  the  specific  gravity  is  an  indication  of  greater  value,  as  it  can 
be  readily  observed  with  proper  appliances.  The  test  has  practical  value,  however, 
only  as  a  confirmation  of  some  other  one.  If  by  the  copper  solutions,  for  instance, 
a  strong  indication  is  obtained  which  it  is  suspected  may  be  due  to  an  excess  of  uric 
acid,  the  reaction  by  fermentation  may  be  resorted  to  because  only  sugar  will 
respond  to  it. 

The  Amount  of  Sugar. — It  is  not  always  sufficient  to  be  able  to  detect  the  pres- 
ence of  sugar  in  urine.  A  knowledge  of  the  amount  is  frequently  of  the  greatest 
importance.  A  number  of  methods  have  been  proposed  by  which  a  quantitative 
determination  can  be  made,  some  of  them  crude  and  of  little  practical  value,  while 
others  give,  when  properly  carried  out,  results  which  are  accurate.  The  methods  in 
general  may  be  divided  into  four  groups,  depending  on  the 

(1)  Reduction  of  solutions  of  heavy  metals,  and  measurement  of  the  amount  of 
reduction. 

(2)  Change  of  color  produced  in  organic  solutions,  by  action  of  sugar,  the  depth 
of  final  color  being  proportional  to  the  amount  of  sugar. 

(3)  Results  of  fermentation,  with  measurement  of  change  in  specific  gravity  of 
the  urine,  or  measurement  of  evolved  carbon  dioxide. 

(4)  Observation  of  rotary  polarization  of  light. 

The  reduction  methods,  which  alone  need  be  considered  here,  are  illustrated  in 
the  use  of  the  Fehling  solution  as  a  qualitative  test  and  in  the  bismuth  tests. 
The  general  principles  involved  in  making  a  quantitative  determination  of  sugar  by 
aid  of  the  Fehling  solution  are  the  same  as  those  involved  in  making  other  volu- 
metric analyses  with  standard  solutions,  and  are  fully  explained  in  Chapter  III. 
A  measured  volume  of  the  properly  prepared  Fehling  solution  is  poured  into  a  flask 
and  brought  to  the  boiling  point.  Then  from  a  burette  the  urine  is  run  in  slowly, 
a  few  cubic  centimeters  at  a  time,  until  the  deep  blue  of  the  copper  solution  is  just 
discharged,  leaving  a  pale  yellow.  At  this  stage  the  copper  is  all  reduced  to  the 
condition  of  insoluble  red  oxide,  Cu20,  and  the  volume  of  urine  added  from  the 
burette  contains  the  amount  of  sugar  measured  by  the  oxidizing  power  of  the 
Fehling  solution  taken.  The  details  of  the  titration  are  as  follows :  Use  the  stand- 
ard quantitative  Fehling  solution  as  described,  and  dilute  an  accurately  measured 
volume  with  exactly  4  volumes  of  water.  That  is,  50  cc.  should  be  diluted  to  250 
cc.  or  25  cc.  to  125.  One  cubic  centimeter  of  this  diluted  liquid  will  oxidize  almost 
exactly  one  milligram  of  glucose,  as  was  shown,  provided  the  sugar  is  in  approxi- 


SOME    PRACTICAL    URINE   TESTS.  321 

mately  i  per  cent,  solution.  For  all  practical  purposes  of  urine  analysis  the  oxidiz- 
ing power  may  be  considered  the  same  in  a  solution  of  one-half  per  cent  strength, 
and  only  very  slightly  increased  in  still  weaker  solutions.  Therefore,  before  begin- 
ning the  test  dilute  the  urine,  if  necessary,  accurately  with  four  or  nine  volumes 
of  water.  This  can  be  done  by  making  50  cc.  up  to  250  or  to  500  cc.  and  mixing 
well  by  shaking. 

With  the  diluted  urine  so  prepared  proceed  as  follows :  Measure  out  50  cc.  of  the 
dilute  Fehling  solution,  pour  it  in  a  flask  and  heat  to  boiling  on  gauze.  Fill  a 
burette  with  the  diluted  urine  and  when  the  solution  in  the  flask  is  actively  boiling 
run  in  about  3  cc.  Boil  two  minutes,  remove  the  lamp,  and  wait  half  a  minute  to 
observe  the  color.  If  blue  is  still  visible,  heat  to  boiling  again  and  run  in  3  cc.  more. 
After  boiling  two  minutes  as  before,  wait  a  short  time  and  observe  the  color  near 
the  surface  of  the  liquid  in  the  flask.  If  still  blue  repeat  these  operations  until 
on  waiting  it  is  found  that  the  blue  has  given  place  to  a  yellow.  The  urine  should 
be  so  dilute  that  at  least  10  cc.  must  be  run  in  to  reduce  all  the  copper  hydroxide. 

When  the  volume  required  is  found  to  within  2  or  3  cc.  a  second  experiment  must 
be  made,  the  urine  being  added  very  gradually  now,  without  interrupting  the  boil- 
ing longer  than  necessary,  until  the  first  of  the  limits  between  which  the  correct 
result  must  lie,  as  shown  by  the  former  test,  is  reached.  From  this  point  the 
addition  of  the  urine  is  continued,  with  frequent  pauses  for  observation  of  color, 
until  the  reduction  is  complete.  The  volume  of  urine  used  contains  50  milligrams 
of  sugar. 

If  the  preliminary  experiment  shows  that  the  urine  is  strong  in  sugar  and  that 
the  reduction  is  easy,  that  is,  that  the  cuprous  oxide  separates  and  settles  readily, 
the  second  test  may  advantageously  be  made  with  50  cc.  of  a  stronger  Fehling  solu- 
tion. With  many  strong  diabetic  urines  it  is  possible  to  use  the  undiluted  copper 
solution  with  the  oxidizing  power  of  4.75  milligrams  of  sugar  to  each  cubic  centi- 
meter. The  difficulties  in  this  test  have  been  very  much  overestimated ;  with  a  little 
practice  any  one  can  make  a  good  sugar  determination  in  urine.  The  important 
point  is  to  find  by  a  few  simple  preliminary  tests  the  best  conditions  of  dilution  of 
Fehling  solution  and  urine  to  give  a  precipitate  which  settles  readily.  With  this 
information,  and  it  can  be  acquired  in  a  few  minutes,  the  actual  quantitative  experi- 
ment can  be  easily  made. 

As  an  illustration  of  the  calculations  involved  let  it  be  assumed  that  50  cc.  of 
the  dilute  Fehling  solution  is  reduced  by  11  cc.  of  urine.  Each  cubic  centimeter 
of  the  urine  must  therefore  contain  4.54  milligrams  of  sugar.  If  the  urine  was 
undiluted  this  corresponds  to  4.54  grams  to  the  liter.  If  it  had  been  diluted  with 
9  volumes  of  water  the  result  must  be  multiplied  by  10,  giving  as  the  original 
strength  45.4  grams  per  liter.  If  the  specific  gravity  of  the  urine  were  found  to  be 
1.032,  the  percentage  strength  would  be 

4-54 
1.032 

Method  by  Use  of  Ammoniacal  Copper  Solution.  This  is  described  fully  in 
Chapter  III.  The  solution  can  be  used  with  dilute  urines  only,  as  it  has  about 
one-fifth  of  the  oxidizing  value  of  the  standard  Fehling  solution.  For  urine  work 
one  cc.  is  considered  the  equivalent  of  one  milligram  of  glucose. 

The  great  advantage  in  the  use  of  this  solution  rests  in  the  fact  that  the  end  point 
is  easily  recognized  by  the  disappearance  of  color  of  the  standard  solution  when 
the  copper  is  all  reduced.  Cuprous  oxide;  dissolves  in  ammonia  without  color  while 
the  slightest  trace  of  cupric  oxide  leaves  a  marked  blue  in  the  liquid.  To  use 
this  solution  with  advantage  the  student  should  learn  to  judge,  from  the  results  of 
22 


322  PHYSIOLOGICAL    CHEMISTRY. 

a  qualitative  test,  about  how  far  the  urine  should  be  diluted.  A  very  strong  dia- 
betic urine  can  be  accurately  titrated  only  after  marked  dilution. 

Other  Sugars  in  Urine.  Besides  the  glucose  other  sugars  are  occasionally  found 
in  the  urine.  Fructose  and  lactose  have  been  frequently  described,  and  also  a 
pentose.  The  identification  of  these  sugars  calls  for  certain  precautions  which  can 
not  be  detailed  here.  The  significance  of  the  pentose  has  been  the  subject  of  much 
discussion.     Fructose  is  found,  as  a  rule,  only  when  glucose  is  present. 

In  addition  to  the  sugars  a  few  other  bodies  are  occasionally  found  in  urine 
which  exhibit  some  of  the  reduction  and  similar  reactions.  One  of  the  most  char- 
acteristic of  these  is  glucoronic  acid,  C6H10O„  which  in  structure  bears  some  rela- 
tion to  glucose.  The  acid  is  a  strong  reducing  agent,  reacts  with  phenylhydrazine 
and  possesses  a  marked  dextro-rotating  power.  When  distilled  with  hydrochloric 
acid  it  yields  f  urf urol,  recognized  by  its  reaction  with  aniline.  In  urine  the  glucoronic 
acid  usually  occurs  in  ester-like  combinations  with  phenols  and  other  bodies. 
These  exhibit  a  levo-rotation,  which  changes  to  the  other  direction  on  boiling  the 
urine  with  a  little  acid.  This  optical  property  is  of  importance  in  the  recognition 
of  the  acid. 

ACETONE,  ACETOACETIC  ACID  AND  OXYBUTYRIC  ACID. 

The  first  of  these  is  frequently  found  in  urine  in  small  amount.  Indeed,  it  may 
be  true,  as  has  been  asserted,  that  it  is  normally  always  present  in  traces.  This 
physiological  acetonuria  has  no  clinical  significance.  Under  some  circumstances, 
however,  it  may  be  found  in  larger  quantity,  sometimes  in  amount  sufficient  to  be 
detected  by  the  odor  alone,  which  fact  first  called  attention  to  it.  At  one  time  it 
was  supposed  to  be  related  to  the  sugar  found  in  urine,  but  it  is  now  established  that 
it  more  generally  accompanies  albumin  and  is  frequently  observed  in  many  febrile 
conditions. 

Acetone  in  urine  is  believed  to  be  a  decomposition  product  of  albumins,  or  of 
bodies  which  may  in  turn  be  looked  upon  as  resulting  from  protein  disintegration, 
the  fatty  acids,  for  example.  It  has  been  shown  that  in  health,  even,  it  can  be 
much  increased  by  a  diet  rich  in  nitrogenous  materials. 

But,  occurring  as  it  does  in  fevers  and  in  advanced  stages  of  diabetes  mellitus, 
a  certain  interest  attaches  to  its  detection,  and  numerous  methods  have  been  pro- 
posed by  which  it  may  be  identified  in  small  amount.  Those  which  depend  on  its 
direct  recognition  in  the  urine  are  mostly  uncertain.  It  is  always  safer  to  distil 
the  liquid  and  apply  the  test  to  a  portion  of  the  distillate.  Half  a  liter,  or  more, 
of  the  urine  is  poured  in  a  retort  attached  to  a  Liebig's  condenser,  and,  after  ad- 
dition of  a  little  phosphoric  acid,  is  subjected  to  distillation,  ioo  cc.  of  distillate 
will  be  enough.     A  portion  of  this  can  be  taken  for  each  test  as  follows : 

Legal's  Test. — Add  to  25  cc.  of  the  liquid  a  small  amount  of  a  fresh  solution  of 
sodium  nitroprusside,  and  a  few  drops  of  a  strong  potassium  hydroxide  solution. 
If  a  ruby-red  color  appears  which  slowly  gives  place  to  yellow,  and  if  the  addition 
of  acetic  acid  changes  this  to  purple  or  violet-red,  the  presence  of  acetone  is 
indicated. 

Creatinine  gives  a  ruby-red  color  as  does  acetone  when  the  nitroprusside  reaction 
is  directly  applied  to  urine,  but  after  adding  acetic  acid  a  green  or  blue  color  results. 

Lieben's  Test. — This  depends  on  the  production  of  iodoform,  and  is  carried  out 
in  this  manner.  To  about  5  cc.  of  the  distillate  add  a  few  drops  of  a  solution 
of  iodine  in  potassium  iodide  (the  "compound  solution  of  iodine,"  Lugol's  solution), 
and  then  enough  sodium  hydroxide  to  make  distinctly  alkaline.  Warm  gently.  If 
acetone  is  present  a  yellowish  white  precipitate  soon  appears,  which,  on  standing, 
becomes  crystalline  and  more  deeply  colored.  The  test  is  said  to  be  sharper  and 
more  characteristic  if  ammonia  is  used  instead  of  the  fixed  alkali.     The  liquid  is 


SOME    PRACTICAL    URINE    TESTS.  323 

first  made  strongly  alkaline  with  ammonia,  and  then  the  iodine  solution  is  added 
until  the  brownish  precipitate  formed  at  first  dissolves  very  slowly.  In  a  short 
time  the  yellowish  iodoform  precipitate  makes  its  appearance.  A  rough  quantita- 
tive measure  of  the  amount  of  acetone  present  is  given  by  noting  the  smallest 
volume  of  the  distillate  with  which  a  distinctive  iodoform  reaction  can  be  seen. 
It  is  said  that  0.0001  milligram  in  1  cc.  can  be  detected;  0.5  milligram  in  10  cc.  can 
be  recognized  by  the  nitroprusside  reaction. 

Acetoacetic  Acid.  This  acid  is  frequently  found  with  acetone  in  the  urine  of 
fevers  and  diabetes,  and  also  in  many  other  pathological  conditions,  but  its  rela- 
tion to  these  disorders  is  not  clearly  understood.  While  acetone  may  be  normally 
present,  the  acetoacetic  acid  is  probably  always  pathological. 

As  it  is  extremely  unstable  tests  for  it  must  be  made  in  the  fresh  urine.  As 
it  yields  acetone  by  decomposition,  tests  for  this  substance  must  be  made  first.  If 
these  are  negative  there  is  no  need  in  going  further,  but  if  they  are  positive  the 
following  direct  test  may  be  made  for  the  acid. 

Ferric  Chloride  Test.  Add  this  reagent,  a  few  drops  at  a  time,  and  look  for 
the  formation  of  a  reddish  color.  Ordinarily  there  can  be  nothing  in  the  urine  to 
give  a  similar  reaction,  and  the  color  test  is  a  strong  indication  of  the  presence  of 
acetoacetic  acid.  But  several  coal  tar  products  given  as  remedies  furnish  residues 
in  the  urine  which  also  give  a  red  or  purple  color  with  ferric  chloride  when  added. 
To  detect  the  acetoacetic  acid  with  certainty  under  these  conditions  it  is  necessary 
to  proceed  with  greater  care.  To  this  end  add  to  the  urine,  which  should  be  fresh, 
a  few  drops  of  ferric  chloride  or  enough  to  precipitate  the  phosphates  present. 
Filter  and  add  a  little  more  of  the  chloride.  A  red  color  indicates  the  acid.  Divide 
the  liquid  into  two  portions ;  boil  one  and  allow  the  other  to  stand  a  day  or  more. 
In  the  boiled  portion  the  color  due  to  acetoacetic  acid  should  disappear  within  a 
few  minutes,  while  in  the  other  it  should  remain  about  twenty-four  hours. 

Acidulate  another  portion  of  the  urine  with  dilute  sulphuric  acid  and  extract  it 
with  ether,  which  takes  up  acetoacetic  acid.  Remove  the  ethereal  layer  and  shake 
it  with  a  very  dilute  aqueous  solution  of  ferric  chloride.  The  red  color  in  the 
new  aqueous  layer  should  appear  as  before  and  disappear  on  boiling,  which  be- 
havior distinguishes  the  acid  from  other  substances  likely  to  be  present. 

^g-Oxybutyric  Acid.  The  detection  of  this  acid  in  the  urine  by  chemical  methods 
is  by  no  means  simple,  but  if  present  in  amounts  not  too  minute  it  may  be  found 
by  the  aid  of  the  polariscope,  inasmuch  as  its  solutions  possess  a  strong  negative 
rotation.  In  dilute  solution  the  specific  rotation  is  approximately  [oi]d  =  —  23.40. 
As  this  acid  is  found  associated  with  sugar  in  diabetes  it  is  necessary  to  destroy  the 
sugar  by  fermentation  before  making  the  test.  Amounts  as  high  as  200  grams  in 
the  day's  urine  have  been  reported,  but  usually,  where  present  at  all,  the  amount  is 
far  below  this,  15  to  20  grams  being  nearer  the  average. 

If  the  urine  does  not  give  a  test  for  acetoacetic  acid  it  is  useless  to  look  for  the 
/3-oxybutyric  acid.  To  test  for  the  latter  evaporate  about  50  cc.  of  the  urine,  freed 
from  sugar  if  necessary,  to  a  syrup  and  distil  the  residue,  mixed  with  an  equal 
volume  of  strong  sulphuric  acid,  from  a  small  flask.  Collect  the  distillate  without 
a  cooler  in  a  test-tube.  If  the  oxybutyric  acid  was  present  in  the  urine  the  dis- 
tillate will  contain  a-crotonic  acid,  which  on  strong  cooling  yields  a  crystal  mass 
melting  at  about  72°  C.  To  get  characteristic  crystals  it  may  be  necessary  to 
extract  the  distillate  with  ether  and  allow  this  to  evaporate. 

NORMAL  COLORING-MATTERS. 

Although  many  investigations  have  been  carried  out  on  the  subject  of  the  normal 
urinary  pigments  we  are  yet  unable  to  give  a  very  definite  account  concerning  them. 
This  is  partly  due  to  the  fact   that  the  coloring  substances  exist  in   the  urine  in 


324  PHYSIOLOGICAL    CHEMISTRY. 

minute  traces  only,  which  makes  their  separation  and  recognition  exceedingly  diffi- 
cult, and  partly  to  another  fact  that  some  of  them  are  easily  altered  or  destroyed 
by  the  action  of  the  reagents  employed  in  their  investigation.  By  proceeding  accord- 
ing to  different  methods,  physiologists  have  obtained  very  different  results  indicating 
the  existence  of  several  colors,  or  at  any  rate  modifications  of  colors.  The  diffi- 
culty of  detecting  the  normal  colors  in  urine  is  sometimes  increased  by  the  pres- 
ence of  traces  of  accidental  coloring-matters  having  their  origin  in  peculiar  or 
unusual  articles  of  food  consumed.     Some  of  these  will  be  referred  to  below. 

It  seems  to  be  settled,  however,  that  in  health  not  merely  one  but  several  coloring- 
bodies  must  be  present.  It  has  not  yet  been  found  possible  to  separate  these  in  the 
free  state. 

Uroerythrin  is  the  name  given  by  Thudichum  and  others  to  a  common  reddish 
coloring-matter  which  often  precipitates  with  urates  and  other  substances.  It  is 
colored  green  by  solution  of  potassium  hydroxide,  but  the  color  is  not  restored  by 
addition  of  acid. 

Urochrome.  This  seems  to  be  the  most  important  and  characteristic  coloring 
matter  of  normal  urine.  It  is  responsible  for  the  ordinary  yellow  color  of  the 
secretion,  and  its  decomposition  products  appear  on  treatment  of  the  urine  with 
strong  acids. 

Urobilin  is  a  coloring  matter  found  in  much  smaller  amount  than  the  last  one. 
When  separated  it  is  a  reddish  brown  amorphous  substance.  In  the  urine  the  fol- 
lowing test  is  sufficient  for  identification.  Precipitate  200  cc.  of  urine  with  basic 
lead  acetate,  collect  the  precipitate  on  a  filter,  wash  it  with  water  and  dry  it,  and 
then  wash  with  alcohol.  Finally,  digest  with  alcohol  containing  a  little  sulphuric 
acid,  and  filter.  The  filtrate  is  usually  fluorescent.  Make  it  strongly  alkaline  with 
ammonia,  and  add  solution  of  zinc  chloride.  This  will  give  the  fluorescence  re- 
ferred to  above  if  but  little  is  added,  while  if  an  excess  of  the  zinc  chloride  is 
added,  a  reddish  precipitate  falls. 

Urophain.  This  is  the  name  given  by  Heller  to  a  substance  identical  with,  or 
similar  to,  urobilin.  Heller  gives  this  test:  Take  a  few  cubic  centimeters  of  strong 
sulphuric  acid  in  a  conical  glass  and  pour  on  it,  drop  by  drop,  about  twice  as  much 
urine.     As  the  two  mix,  a  deep  garnet-red  is  produced. 

This  reaction  is  not,  however,  characteristic,  as  several  other  matters  may  give  it. 

Urohematin  is  the  name  given  by  Harley  to  a  coloring-matter  similar  to  the 
above.  He  applies  this  test:  Dilute  or  concentrate  the  urine  so  that  it  is  equivalent 
to  1,800  cc.  for  the  twenty-four  hours.  Take  a  few  cubic  centimeters  in  a  test-tube 
or  wine-glass,  and  add  one-fourth  of  its  volume  of  strong  nitric  acid.  No  change 
of  color  can  be  observed  if  the  urohematin  is  present  in  normal  amount,  but  if 
in  excess  various  shades  from  pink  to  red  may  appear. 

Indican.  This  is  a  normal  constituent  of  urine  in  small  amount,  but  in  many 
intestinal  diseases  may  be  greatly  increased.  Its  detection  in  the  urine  is  then  a 
matter  of  considerable  clinical  importance.  It  is  formed  from  the  oxidation  of 
indol  which  is  a  common  product  of  bacterial  origin  in  the  intestines.  The  oxida- 
tion product,  indoxyl,  combines  with  sulphuric  acid  and  the  potassium  salt  of  this 
is  excreted  as  indican.  The  formula  of  the  indican  is  KS04C8H6N.  Through  fur- 
ther oxidation  this  yields  indigotin,  a  blue  coloring-matter  which  is  the  substance 
finally  identified  in  the  test.     For  further  relations  consult  earlier  chapters. 

Indican  is  found  in  normal  urines  in  very  small  amount  only.  It  may,  under 
favorable  circumstances,  be  detected  as  here  given :  Take  about  4  cc.  of  pure  hydro- 
chloric acid  in  a  test-tube  and  add  about  half  as  much  urine,  shaking  well.  A 
blue  or  violet  color  shows  indican.  This  test  depends  on  the  conversion  of  the 
indoxyl  compound  into  indigo,  but  the  oxidizing  action  of  the  acid  is  not  always 
strong  enough  to  bring  about  the  change  in  presence  of  other  organic  bodies  in  the 
urine. 


SOME    PRACTICAL    URINE    TESTS.  325 

A  more  generally  applicable  method  is  this :  To  10  cc.  of  urine  and  the  same 
volume  of  strong  pure  hydrochloric  acid,  add  2  or  3  cc.  of  chloroform.  Then 
add,  drop  by  drop,  solution  of  sodium  hypochlorite,  shaking  after  each  addition. 
The  hypochlorite  acts  as  an  oxidizing  agent,  liberating  the  coloring-matter,  which 
is  then  taken  up  by  the  chloroform.  The  oxidation  must  not  be  carried  too  far; 
that  is,  too  much  hypochlorite  must  not  be  added,  as  it  would  then  destroy  the  color 
as  fast  as  formed.  In  fact,  small  traces  of  the  product  sought  might  be  completely 
overlooked  in  the  process,  as  the  hypochlorite  is  a  very  active  oxidizer,  the  effect 
going  far  beyond  the  production  of  indigo.  It  has  therefore  been  proposed  to  use 
nitric  acid  as  the  oxidizing  agent.  The  urine  is  boiled  with  an  equal  volume  of 
hydrochloric  acid  and  then  a  few  drops  of  nitric  acid  are  added.  The  mixture  is 
cooled  and  to  it  a  little  chloroform  is  added  and  well  shaken.  In  presence  of  indigo 
the  blue  color  appears.  Bromine  water  in  small  amount  may  be  employed  in  the 
same  manner. 

More  recently  hydrochloric  acid  containing  a  little  ferric  chloride  has  come  into 
use  as  an  oxidizing  agent.  This  is  less  destructive  than  the  nitric  acid  or  hypo- 
chlorite. The  reagent  may  be  made  by  dissolving  3  or  4  grams  of  the  ferric  chloride 
in  a  liter  of  strong  hydrochloric  acid.     It  is  known  as  the  Obermeyer  reagent. 

As  a  comparative  quantitative  test  the  following  may  be  employed :  To  a  hundredth 
part  of  the  whole  day's  excretion  add  an  equal  volume  of  the  above  reagent  and 
5  cc.  of  chloroform.  Shake  a  minute  or  more,  and  allow  to  settle.  Compare  the 
color  with  that  of  a  strong  Fehling  solution,  taken  as  100,  as  a  standard. 

ABNORMAL  COLORS. 

We  have  here  a  number  of  pathological  substances  which  are  best  recognized 
through  certain  color  reactions.  These  substances  may  not  have  much  significance 
in  themselves  but  they  are  often  important  in  pointing  to  certain  conditions,  indi- 
rectly. 

The- Bile  Pigments.  Several  coloring-matters  originating  in  the  liver  may  find 
their  way  into  the  urine.  Their  recognition  is  frequently  important,  and  several 
reactions  are  available  for  this. 

Biliary  urine  has  generally  a  characteristic  greenish  yellow  color  sometimes  tinged 
with  brown.  The  froth  from  such  urine  is  readily  recognized  by  its  yellow  color, 
which  is  often  a  sufficient  test  in  itself.  Among  the  chemical  tests  the  following  are 
the  best  known. 

Gmelin's  Test.  This  is  easily  performed  and  depends  on  the  oxidation  of  bili- 
rubin, the  pigment  commonly  present  in  fresh  jaundice  urine,  by  nitrous  acid.  Pour 
in  a  test-tube  about  5  cc.  of  the  urine  under  examination  and  by  means  of  a  pipette 
introduce  below  it  an  equal  volume  of  strong  nitric  acid  mixed  with  nitrous.  This 
should  be  carefully  done  so  as  to  avoid  mixing  the  liquids  much.  At  the  junction 
of  the  two  liquids,  if  bile  is  present,  several  colored  rings  appear  of  which  the  green 
due  to  biliverdin  is  most  characteristic.  The  bands  or  rings  appear  above  the  acid 
in  this  order,  yellowish  red,  red,  violet,  blue,  and  green.  The  last  is  essential.  It 
must  be  remembered  that  nitric  acid  gives  the  other  colors  at  times  with  urine  free 
from  bile,  but  green  is  characteristic  of  the  latter. 

Fleischl  modified  this  test  by  mixing  the  urine  with  a  strong  solution  of  sodium 
nitrate  and  then  adding  strong  sulphuric  acid  carefully.  This  settles  below  the 
urine  and  decomposes  the  nitrate  at  the  point  of  contact,  liberating  the  necessary 
nitric  and  nitrous  acids  for  the  oxidation  as  before.    This  method  is  a  very  good  one. 

Trousseau's  Test.  Add  to  some  urine  in  a  test-tube  a  few  drops  of  tincture 
of  iodine,  allowing  this  to  float  over  the  urine.  If  the  bile  pigments  are  present  a 
greenish  color  appear!  at  the  junction  of  the  two  liquids  and  may  remain  some 
hours.     An  excess  of  iodine  must  not  be  used. 


326  PHYSIOLOGICAL    CHEMISTRY. 

The  Diazo  Reaction.  Ehrlich  and  others  have  called  attention  to  the  behavior 
of  many  urines  with  solution  of  diazobenzene  sulphonic  acid,  which  often  has  im- 
portance in  diagnosis.  Normal  urine  treated  with  a  weak  solution  of  this  reagent 
shows  no  marked  change,  but  in  several  pathological  conditions  after  adding  the 
acid  and  saturating  with  ammonia  a  deep  carmine  or  scarlet-red  color  appears,  fol- 
lowed by  greenish  or  violet  precipitation.  After  the  addition  of  ammonia  the  foam 
should  show  a  distinct  pink  color. 

This  reaction  depends  on  the  combination  of  the  sulphonic  acid  of  diazobenzene 
with  some  aromatic  compound  found  in  the  urine  in  pathological  condition.  At  one 
time  it  was  supposed  to  have  special  significance  in  the  diagnosis  of  typhoid  fever, 
but  it  now  appears  that  in  many  diseases  of  the  intestinal  tract  the  urine  receives 
traces  of  complex  aromatic  products  of  bacterial  origin  which  respond  to  the  test. 
It  has,  therefore,  general  rather  than  special  significance. 

As  the  diazobenzene  sulphonic  acid  is  not  very  stable,  it  is  not  convenient  to  use, 
and  a  reagent  is  made  which,  in  its  application,  is  its  chemical  equivalent.  The 
reagent  is  prepared  by  dissolving  1  gram  of  sulphanilic  acid  in  200  cc.  of  water 
with  the  addition  of  10  cc.  of  pure  hydrochloric  acid.  Another  solution  is  made  by 
dissolving  1  gram  of  sodium  nitrite  in  200  cc.  of  water.  To  make  the  test  take  50 
cc.  of  the  first  solution,  add  5  cc.  of  the  nitrite  solution  and  then  50  cc.  of  urine. 
Ammonia  is  then  added  in  sufficient  quantity  to  impart  a  strong  alkaline  reaction 
after  thoroughly  shaking.  A  scarlet-red  color  is  the  result  if  the  urine  in  question 
contains  the  abnormal  products  referred  to. 

It  must  be  remembered  that  normal  urines  usually  give  some  color.  It  is  only 
the  strong  reactions  which  have  significance. 

Ehrlich  has  introduced  another  test  which  discloses  the  presence  of  products  of 
intestinal  origin.  This  is  a  2  per  cent  solution  of  dimethyl  amino  benzaldehyde  in 
15  per  cent  hydrochloric  acid.  A  few  drops  of  this  reagent  added  to  5  cc.  of  urine 
strikes  a  red  color,  weak  in  normal  urine,  but  deep  in  certain  pathological  urines. 
The  exact  significance  of  the  reaction  is  not  known. 

BLOOD  COLORING  MATTERS. 

These  may  appear  in  the  urine  from  several  sources.  We  may  have  color  due  to 
the  presence  of  corpuscles  themselves  and  we  may  have  the  color  due  to  dissolved 
hemoglobin.  It  is  important  to  distinguish  between  these  conditions.  The  presence 
of  the  corpuscles,  which  are  best  recognized  by  the  microscope,  points  to  a  lesion  of 
the  kidney  or  some  part  of  the  urinary  tract,  and  to  a  fresh  lesion  if  the  corpuscles 
are  sharp  in  outline.  The  term  hematuria  is  applied  to  the  condition  when  cor- 
puscles themselves  are  present,  while  hemoglobinuria  is  the  condition  in  which  few 
or  no  corpuscles  are  found,  but  the  free  pigment  is  present.  Hemoglobinuria  is  the 
result  of  the  disintegration  of  corpuscles  within  the  tissues  or  blood  vessels,  per- 
mitting the  oxyhemoglobin  or  some  derivative  to  pass  through  the  kidney  with  the 
urine.  With  much  of  the  coloring  matter  present,  the  urine  may  have  a  very  dark 
color.  Sometimes  the  hemoglobin  suffers  further  decomposition,  giving  rise  to 
hematin  and  methemoglobin. 

The  following  are  the  best  chemical  tests  'for  the  recognition  of  these  bodies : 

Heller's  Test.  Treat  the  urine  with  solution  of  sodium  or  potassium  hydrox- 
ide, and  heat  to  boiling.  This  produces  a  precipitate  of  the  earthy  phosphates  which 
in  subsiding  carry  down  coloring-matters.  If  a  precipitate  does  not  separate  readily 
it  may  be  hastened  by  adding  two  or  three  drops  of  magnesia  mixture.  Hemo- 
globin, when  present,  is  decomposed  by  this  treatment  with  separation  of  hematin, 
which  in  turn  settles  down  with  the  phosphates,  imparting  a  red  color  to  the 
precipitate. 

Struve's  Test.     Make  the  urine  slightly  alkaline  with  sodium  hydroxide  solu- 


SOME    PRACTICAL    URINE    TESTS.  327 

tion,  and  then  add  enough  solution  of  tannic  acid  in  acetic  acid  to  change  the  re- 
action. If  hemoglobin  is  present  a  dark  brown  precipitate  of  hematin  tannate 
settles  out.  The  test  is  a  good  one,  and  easily  performed,  but  is  not  sufficiently 
delicate  for  the  detection  of  small  traces  of  hemoglobin  directly.  By  collecting 
the  precipitate  on  a  filter  and  washing  it,  it  may  be  used  for  two  confirmatory  tests. 
One  of  these  depends  on  the  formation  of  hemin  crystals  and  is  made  in  this 
manner:  Place  a  small  portion  of  the  precipitate  on  a  microscopic  glass  slide  and 
add  a  minute  crystal  of  sodium  chloride.  Then  add  a  large  drop  of  glacial  acetic 
acid  and  cover  with  a  cover  glass.  Warm  very  gently  over  a  small  flame.  When 
the  acid,  salt,  and  precipitate  have  become  thoroughly  mixed  allow  the  slide  to  cool. 
Small  rhombic  crystals  of  hemin  should  now  appear,  which  are  best  seen  under  a 
microscope. 

The  washed  precipitate  may  also  be  ashed  and  used  for  an  iron  test.  The  ash 
should  be  dissolved  in  a  little  pure  hydrochloric  acid  in  a  porcelain  dish  and  tested 
by  the  addition  of  potassium  ferrocyanide  and  ferricyanide  to  give  the  well-known 
reaction.  This  test  presupposes  purity  and  freedom  from  traces  of  iron  in  the 
reagents  used. 

Almen's  Guaiacum  Test.  In  a  test-tube  mix  equal  volumes  of  fresh  tincture 
of  guaiacum  and  ozonized  turpentine — 2  or  3  cc.  of  each  will  suffice.  The  mixture, 
if  made  of  proper  materials,  must  not  show  a  green  or  blue  color  after  thoroughly 
shaking.  Now  add  a  few  cubic  centimeters  of  the  urine  to  be  tested,  a  drop  at  a 
time,  and  agitate  after  each  addition.  If  hemoglobin  is  present  it  causes  the  oxidiz- 
ing material  of  the  ozonized  turpentine  (probably  hydrogen  peroxide)  to  act  on  the 
precipitated  guaiacum  resin,  imparting  to  it  first  a  greenish,  and  finally  a  blue  color. 
Old  and  alkaline  urine  must  be  made  faintly  acid  before  performing  the  test.  Pus 
in  the  urine  gives  a  somewhat  similar  reaction,  and  a  few  other  bodies,  very  seldom 
present,  interfere.  The  test  is  very  delicate,  and  if  it  gives  a  negative  result  it  is 
safe  to  conclude  that  blood  is  absent. 

Vegetable  and  Other  Colors.  It  has  long  been  known  that  many  peculiar  col- 
oring-matters enter  the  urine  from  substances  taken  as  remedies,  or  with  the  food. 
Santonin  from  wormseed,  chrysophanic  acid  from  some  kinds  of  rhubarb,  the 
coloring  substances  from  blueberries,  carrots  and  other  fruits  and  vegetables,  are 
all  occasionally  met  with  in  the  urine,  where  they  may  lead  to  confusion.  In  most 
cases  the  addition  of  acid  produces  a  yellowish  tinge,  while  alkalies  give  rise  to  a 
red  color. 

Many  aromatic  compounds  given  as  remedies  pass  into  the  urine  in  small  amount, 
where  they  may  often  be  recognized  by  the  peculiar  color  reactions  they  yield  with 
certain  reagents.  Salicylic  acid  is  an  illustration,  and  it  may  be  recognized  by  the 
blue  color  it  strikes  on  addition  of  ferric  chloride. 

TOTAL  NITROGEN. 

In  all  complete  examinations  of  the  urine  a  determination  of  the  total  nitrogen 
present  is  necessary.  This  may  be  easily  made  by  means  of  the  simple  Kjeldahl 
method,  as  follows : 

Measure  accurately  5  cc.  of  urine  into  a  large  Kjeldahl  digesting  flask  and  add 
25  cc.  of  pure  sulphuric  acid  and  10  grams  of  pure  potassium  sulphate.  Heat  over 
a  free  flame  about  an  hour,  or  until  the  mixture  has  become  perfectly  colorless. 
Complete  hydrolysis  is  effected  and  the  nitrogen  is  all  left  as  ammonium  sulphate. 
Cool  the  flask,  dilute  largely  with  distilled  water,  neutralize  with  an  excess  of  pure 
alkali  solution  and  distill  off  the  ammonia. 

This  ammonia  is  caught  in  25  cc.  of  N/4  sulphuric  acid,  which  is  titrated  after- 
wards with  corresponding  alkali,  using  alizarin  red  or  methyl  orange  as  indicator. 
The  results  are  accurate. 


328  PHYSIOLOGICAL   CHEMISTRY. 

The  total  nitrogen  determination  should  always  be  made  as  a  check  on  the  sum 
of  the  nitrogen  factors  found  in  the  following  paragraphs.  It  will  be  remembered 
that  the  urea  nitrogen  makes  up  a  large  fraction  of  the  total  nitrogen. 

UREA. 

The  physiological  importance  of  urea  was  discussed  in  the  last  chapter.  As  it 
makes  up  a  large  fraction  of  the  nitrogen  excretion,  which  in  turn  varies  with  the 
protein  of  the  diet,  the  total  amount  excreted  may  vary  between  a  few  grams  and 
fifty  grams  daily.  By  bacterial  agency  it  is  rather  quickly  converted  into  ammonium 
carbonate  in  the  voided  urine.  Quantitative  tests  should  be  made,  therefore,  on 
fresh  urine  only. 

Recognition  of  Urea.  Because  of  its  extreme  solubility  urea  cannot  be  easily 
obtained  by  evaporation  of  urine.  It  may  be  shown,  however,  by  a  simple  experi- 
ment that  by  concentrating  the  urine  slowly  to  a  small  volume — to  one-third  or 
one-fourth — cooling  and  adding  strong  nitric  acid,  a  crystalline  precipitate  of  plates 
of  urea  nitrate  separates,  which  is  characteristic.  From  this  precipitate  pure  urea 
can  be  obtained. 

Clinically,  this  test  has  no  importance,  as  we  are  concerned  only  with  a  measure- 
ment of  the  amount  of  urea.  This  determination  can  be  made  in  several  ways, 
but  in  actual  practice  we  employ  three  essentially  different  methods.  The  first 
depends  on  the  fact  that  solutions  of  urea  precipitate  solutions  of  certain  metals  in 
a  definite  manner,  from  which  a  volumetric  process  has  been  derived.  The  second 
depends  on  the  fact  that  solutions  of  certain  oxidizing  agents  decompose  solutions 
of  urea  with  the  liberation  of  its  nitrogen  (and  carbon  dioxide)  in  gaseous  form. 
From  the  known  relations  between  weight  and  volume  of  the  gas,  and  weight  of 
nitrogen  and  weight  of  urea,  the  absolute  amount  of  the  latter  may  be  calculated. 
The  third  method  depends  on  the  fact  that  when  the  urea  of  urine  is  heated  in 
solution  under  certain  conditions  it  is  converted  quantitatively  into  ammonium  salts 
from  which  the  ammonia  may  be  distilled  and  measured. 

In  the  practice  of  the  first  method  a  standard  solution  of  mercuric  nitrate  is 
employed,  as  this  precipitates  urea  quantitatively,  but  as  the  reaction  is  complicated 
by  the  presence  of  other  bodies  it  is  seldom  used  practically  at  this  time.  This  is 
the  old  Liebig  method.  Ordinarily  for  clinical  purposes  we  measure  urea  by  the 
second  process;  that  is  from  the  nitrogen  liberated  in  some  oxidation  reaction,  as 
will  now  be  described. 

Method  by  Liberation  of  Nitrogen.  A  solution  of  urea  is  decomposed  by  a 
solution  of  a  hypochlorite  or  hypobromite  as  illustrated  by  this  equation. 

CON,H4  +  3NaOCl  =  C02  +  N2  +  2H20  +  3NaCl. 

That  is,  nitrogen  and  carbon  dioxide  gases  are  given  off.  If  the  reaction  is 
allowed  to  take  place  in  an  alkaline  medium  the  carbon  dioxide  will  be  held  and 
the  nitrogen  alone  given  off.  The  volume  liberated  is  a  measure  of  the  weight  of 
urea  decomposed.  From  the  above  equation  it  is  seen  that  28  parts  by  weight  of 
nitrogen  correspond  to  60  of  urea,  from  which  it  follows  that  1  cc.  of  pure  nitro- 
gen gas,  measured  at  a  temperature  of  o°  C.  and  under  the  normal  pressure  of  760 
mm.  corresponds  to  0.00269  gram  of  urea.  One  gram  of  urea  furnishes  371.4  cc. 
of  nitrogen  gas. 

In  employing  these  principles  in  practice  it  is  simply  necessary  to  bring  together 
a  measured  volume  of  the  urine  or  urea  solution  and  the  hypochlorite  or  hypo- 
bromite reagent  under  such  conditions  that  all  of  the  nitrogen  liberated  may  be 
collected  and  accurately  measured. 

As  a  reagent,  a  solution  of  sodium  hypobromite  is  very  commonly  employed. 
As  it  does  not  keep  well   it  must  be  made  fresh  for  use,  which  is  inconvenient 


SOME    PRACTICAL    URINE    TESTS. 


329 


unless  many  tests  have  to  be  made  at  one  time.  The  reagent  may  be  prepared 
in  this  manner: 

Dissolve  100  grams  of  good  sodium  hydroxide  in  250  cc.  of  water.  When  cold 
add  25  cc.  of  bromine  by  means  of  a  funnel  tube  carried  to  the  center  of  the  solu- 
tion. The  bromine  must  be  poured  into  the  funnel  a  little  at  a  time,  with  constant 
agitation,  the  containing  vessel  being  kept  cold.  There  is  an  excess  of  alkali 
here  to  hold  the  carbon  dioxide  liberated. 

A  very  convenient  form  of  apparatus  used  in  this  test  is  shown  in  the  annexed 
cut. 

The  tall  jar  is  filled  with  water  which  must  stand  until  it  has  the  air  tempera- 
ture. A  50  cc.  burette  is  inverted  in  the  jar,  the 
delivery  end  being  connected  with  a  bottle  holding 
about  150  cc,  by  means  of  a  piece  of  firm  rubber 
tubing.  The  rubber  tube  is  slipped  over  a  short 
glass  tube  passing  through  the  hole  in  a  rubber 
stopper  which  must  close  the  bottle  accurately. 
In  the  bottle  is  a  short  stout  test-tube,  or  vial, 
holding  about  10  cc.  and  which  contains  the  urine 
to  be  tested.  Into  the  bottle  itself  is  poured  the 
reagent  as  above  described,  which  must  not  reach 
to  the  top  of  the  test-tube.  On  mixing  the  liquids 
the  urea  decomposes,  liberating  the  gas  as  ex- 
plained, which  passes  through  the  rubber  tube  and 
displaces  water  in  the  burette  so  that  the  volume 
can  be  readily  determined. 

The  test  is  made  practically  in  this  manner : 
Pour  about  20  cc.  of  the  strong  hypobromite  or  40 
cc.  of  the  weaker  hypochlorite  reagent  into  the 
bottle.  With  a  pipette  measure  some  exact  vol- 
ume of  urine,  usually  5  cc,  into  the  small  test- 
tube,  and  by  means  of  small  iron  forceps  place 
the  latter  carefully  in  the  bottle  containing  the 
reagent.  Insert  the  stopper  which  connects  the 
bottle  with  the  burette  standing  in  the  jar  of 
water.  Allow  the  apparatus  to  stand  about  ten 
minutes  to  get  the  proper  air  temperature,  and 
then  adjust  the  levels  of  the  liquid  in  the  burette 
and  jar.     Note  the  reading  in  the  burette,  and  the 

temperature.  Incline  the  bottle  to  mix  the  urine  and  the  reagent,  and  shake  to  com- 
plete the  reaction.  The  liberated  nitrogen,  or  its  equivalent  volume  of  air,  passes 
over  into  the  burette  and  depresses  the  water.  When  there  is  no  change  in  the  gas 
volume  allow  the  whole  apparatus  to  stand  until  the  contents  of  the  bottle  and 
burette  have  cooled  down  to  the  air  temperature  again.  Then  lift  the  burette, 
with  the  clamp  as  before,  to  restore  the  levels  and  read  the  gas  volume.  From  this 
subtract  the  volume  at  the  first  reading.  The  difference  is  the  volume  of  nitro- 
gen gas  liberated  in  the  reaction,  at  the  observed  temperature  and  atmospheric 
pressure.  If,  as  sometimes  happens,  more  gas  is  liberated  than  the  burette  will 
hold,  repeat  the  experiment,  using  urine  diluted  with  an  equal  volume  of  water. 

Reduce  the  gas  volume  to  standard  conditions  by  the  usual  formulas,  and  for 
each  cubic  centimeter  calculate  2.7  milligrams  of  urea  as  present  in  the  volume 
taken. 

Exact  investigations  have  shown  that  the  whole  of  the  nitrogen  is  not  liberated 
in  the  reaction,  as  at  first  assumed,  but   falls  short  between  7  and  8  per  cent.     It 


330  PHYSIOLOGICAL    CHEMISTRY. 

appears  that  under  some  conditions  a  small  part,  possibly  3  or  4  per  cent.,  of  the 
nitrogen  of  the  urea  is  oxidized  to  nitric  acid  in  the  reaction  and  escapes  meas- 
urement. Another  small  portion  is  left  in  the  ammoniacal  condition.  Attempts 
have  been  made  to  prevent  this  abnormal  oxidation  by  adding  to  the  urine  a  re- 
ducing agent  to  destroy  nitric  acid,  as  fast  as  formed.  Dextrose  has  been  used 
for  the  purpose,  also  cane-sugar,  and  apparently  with  success.  But  a  great  excess 
of  sugar  must  be  added.     The  results  are  therefore  only  approximately  correct. 

Doremus  has  introduced  a  small  apparatus  for  the  quick  and  approximate  esti- 
mation of  urea  in  which  1  cc.  of  urine  is  used.  This  apparatus  is  often  employed 
clinically.     The  same  reagent  is  used. 

In  the  more  exact  methods  of  estimation  ammonia  is  formed  by  hydrolysis  re- 
actions, usually  aided  by  hydrochloric  acid.  Folin  has  simplified  these  methods 
greatly. 

The  Folin  Method.  This  conversion  is  accomplished  by  boiling  the  urine  in  a 
flask  with  magnesium  chloride  and  hydrochloric  acid,  the  boiling  point  of  the  mix- 
ture being  high  enough  to  effect  the  hydration.  The  determination  is  carried  out 
practically  as  follows :  Measure  accurately  5  cc.  of  urine  into  a  200  cc.  Erlenmeyer 
flask,  add  5  cc.  of  strong  hydrochloric  acid  and  20  gm.  of  crystallized  magnesium 
chloride.  Add  also  a  small  piece  of  paraffin,  about  half  a  gram,  and  a  few  drops 
of  alizarin  red  as  indicator.  Attach  a  small  reflux  condenser  to  the  flask,  or  the 
special  condensing  bulb  tube,  which  Folin  recommends,  and  heat  to  boiling.  Con- 
tinue the  ebullition  until  the  drops  returning  from  the  condenser  produce  a  sharp 
sound  when  they  strike  the  liquid,  now  concentrated,  remaining  in  the  flask.  After 
this  the  boiling  may  be  continued  more  gently  through  45  minutes  or  an  hour.  But 
the  reaction  in  the  flask  must  remain  strongly  acid,  as  shown  by  the  indicator.  The 
condensing  bulb  permits  the  return  of  evaporated  acid,  as  necessary.  Then  the 
mixture  is  diluted  with  water,  washed  into  a  liter  flask  with  water  enough  to  give 
a  volume  of  700  cc,  made  alkaline  with  10  cc.  of  20  per  cent,  sodium  hydroxide  and 
distilled.  This  carries  over  the  ammonia  which  is  best  caught  in  N/10  sulphuric 
acid,  of  which  about  50  cc.  should  be  taken.  At  the  end  of  the  distillation,  which  is 
carried  on  nearly  to  dryness,  the  excess  of  acid  is  titrated  back,  and  the  difference 
corresponds  to  the  ammonia.  For  17  milligrams  of  ammonia  calculate  30  milli- 
grams of  urea. 

As  urine  contains  a  little  ammonia,  this  must  be  deducted  in  the  final  cal- 
culation. It  should  be  determined  by  a  special  process.  The  distillation  of  the 
urea  ammonia  requires  about  an  hour  and  in  the  final  titration  alizarin  red  is  used 
as  indicator.  Uric  acid  and  creatinine  are  not  appreciably  attacked  in  this  hydroly- 
sis. With  practice  the  method  gives  very  good  results.  As  much  of  the  mag- 
nesium chloride  on  the  market  contains  traces  of  ammonia  this  must  be  separately 
determined  and  subtracted  from  the  amount  found. 

Benedict  and  Gephart  have  recently  recommended  a  method  in  which  the  hydroly- 
sis of  the  urea  is  effected  by  heating  the  urine  with  dilute  hydrochloric  acid  in  an 
autoclave.  5  cc.  of  urine  is  mixed  with  5  cc.  of  8  to  10  per  cent  acid  in  a  test- 
tube,  which  is  heated  an  hour  and  a  half  to  about  150°  in  the  autoclave.  Many 
tubes  may  be  heated  at  one  time.  The  results  are  a  trifle  higher  than  in  the 
Folin  method,  as  other  constituents  appear  to  be  slightly  hydrolyzed.  The  contents 
of  the  test-tubes  are  diluted  to  about  400  cc,  made  alkaline,  and  distilled  as  in  the 
Folin  method. 

AMMONIA  IN  URINE. 

The  normal  amount  excreted  varies  usually  between  500  milligrams  and  one  gram 
daily,  and  makes  up  about  5  per  cent  of  the  total  nitrogenous  excretion  as  shown 
in  the  last  chapter  where,  also,  the  conditions  of  formation  are  explained. 

Determination  of  Ammonia.    As  ammonia  is  always  present  in  urine  in  some 


SOME    PRACTICAL    URINE    TESTS.  331 

amount,  qualitative  tests  have  little  value,  and  we  proceed  immediately  to  quanti- 
tative methods.  As  the  ammonia  present  is  in  combination  as  a  salt  it  must  be 
liberated  by  the  action  of  a  strong  alkali,  but  in  the  choice  of  one  for  this  pur- 
pose we  are  limited  by  the  fact  that  the  hydroxides  of  sodium  and  potassium 
have  a  decomposing  action  on  urea  and  other  nitrogenous  bodies  in  urine  and 
cannot,  therefore,  be  used.  Milk  of  lime  is  free  from  this  objection  and  may  be 
employed.  The  experiment  is  so  arranged  that  the  liberated  ammonia  may  be 
absorbed  by  a  measured  volume  of  standard  sulphuric  acid,  the  amount  of  this 
neutralized  being  the  measure  of  the  ammonia  absorbed: 

H2S04  +  2NH3=  (NH4)2S04. 

98  parts  by  weight  of  the  acid  correspond  to  34  parts  of  ammonia,  NH3.  The 
old  Schloesing  method  is  based  on  these  principles.  But  it  is  by  no  means  as 
accurate  or  convenient  as  the  later  processes  in  which  the  ammonia  is  drawn  out 
from  a  measured  volume  of  urine  by  an  air  current  or  through  the  aid  of  a  vacuum 
pump,  absorbed  in  standard  acid,  and  so  titrated.  One  of  the  best  of  these  absorp- 
tion processes  is  the  following. 

The  Folin  Ammonia  Determination.  In  this  method  25  cc.  of  urine  is  meas- 
ured into  a  tall  narrow  cylinder  with  about  a  gram  of  dry  sodium  carbonate. 
Enough  light  petroleum  is  poured  into  the  cylinder  to  form  a  layer  about  5  mm. 
deep.  This  is  to  diminish  the  frothing  in  the  following  operation.  The  tall 
cylinder,  furnished  with  a  doubly  perforated  stopper,  is  connected  with  an  absorp- 
tion apparatus  to  wash  the  air  current  to  be  drawn  through,  and  free  it  from 
ammonia,  and  is  followed  by  another  absorption  apparatus  containing  a  measured 
volume  of  N/10  sulphuric  acid.  The  system  is  connected  with  a  large  Chapman 
pump,  capable  of  drawing  several  hundred  liters  of  air  through  in  an  hour.  With 
a  sufficiently  rapid  air  current  bubbling  through  the  urine,  the  ammonia  may  be 
completely  drawn  out  in  an  hour  and  a  half  or  two  hours,  and  absorbed  in  the 
standard  acid,  of  which  25  cc.  should  be  taken  and  diluted  to  200  cc.  with  water. 
To  aid  in  the  absorption  of  the  ammonia  carried  along  by  the  air  current  Folin 
suggests  a  special  bulb  tube  through  which  the  air  must  pass  into  the  standard  acid. 
It  is  also  well  to  put  a  Reitmair  or  Hopkins  bulb  over  the  tall  cylinder  holding 
the  urine,  to  catch  any  liquid  which  may  be  carried  up  by  the  froth  produced  by  the 
rapid  air  current.  The  time  required  in  the  determination  depends  on  the  air  cur- 
rent available,  and  this  should  be  found  by  a  few  preliminary  experiments.  The 
results  are  extremely  exact. 

With  a  good  air  current  it  is  possible  to  work  six  or  eight  of  these  combinations 
in  series,  and  complete  the  tests  in  the  same  time.  One  protecting  washing  flask 
at  the  end  of  the  series  is  sufficient  for  the  whole,  and  the  tube  from  this  leads  to 
the  bottom  of  the  liquid  in  the  first  urine  cylinder.  The  cylinder  may  be  30  to 
36  cm.  high  and  4  or  5  cm.  wide,  with  advantage. 

URIC  ACID. 

The  relations  of  uric  acid  to  the  whole  nitrogen  excretion  were  explained  in 
the  last  chapter.    The  normal  variations  are  between  about  0.2  gm.  and  1.2  gm.  daily. 

In  the  recognition  of  the  acid  the  following  points  may  be  noted:  When  present 
in  large  amount  it  frequently  precipitates  from  the  urine  in  free  form,  or  as  an  acid 
urate ;  in  both  cases  the  precipitate  is  yellow.  When  the  amount  present  is  small  it 
may  be  found  by  acidifying  with  hydrochloric  acid  and  then  allowing  the  urine  to 
stand  some  hours  in  a  cool  place;  uric  acid  crystals  separate.  In  mixed  sediments 
it  may  \><-.  recognized  by  this  test: 

Murexid  Test.  Throw  the  sediment  on  a  filter  and  wash  once  with  water. 
Place  the  residue  in  a  porcelain  dish,  add  a  drop  of  strong  nitric  acid,  and  evaporate 


332  PHYSIOLOGICAL    CHEMISTRY. 

to   dryness    on   the   water-bath.     A  yellow   or  brown  mass   is   obtained,   and   this 
touched  with  a  drop  of  ammonia  water  turns  purple. 

Unless  the  uric  acid  or  urate  is  present  in  the  sediment  in  fine  granular  form 
its  recognition  by  the  microscope  is  very  simple.  Illustrations  of  the  forms  of  uric 
acid  and  certain  urates  are  given  in  the  paragraphs  on  the  sediments. 

THE  AMOUNT  OF  URIC  ACID. 

For  determination  of  the  amount  of  the  acid  in  the  urine  we  have  the  choice 
of  several  methods,  not  one  of  which  is  very  convenient  or  of  the  greatest  accuracy. 
The  first  of  these  depends  on  the  fact  referred  to  above,  that  hydrochloric  acid 
liberates  uric  acid  from  its  combination,  precipitating  it  in  crystalline  form. 

Precipitation  Test.  Measure  out  200  cc.  of  urine  and  add  to  it  20  cc.  of  strong 
hydrochloric  acid.  Mix  thoroughly  and  set  aside  in  a  cool  place  for  about  forty- 
eight  hours.  At  the  end  of  this  time  collect  the  reddish-yellow  deposit  on  a 
weighed  filter,  wash  it  with  a  little  cold  water,  dry,  and  weigh.  Not  over  30  or  40 
cc.  of  water  should  be  used  in  the  washing.  The  precipitated  uric  acid  is  not  pure, 
holding  coloring  and  other  substances  which  increase  its  weight.  On  the  other 
hand,  it  is  soluble  to  some  extent  in  cold  acidulated  water  so  that  not  the  whole 
of  it  is  obtained  on  the  filter  and  a  correction  must  be  made.  It  is  usually  recom- 
mended to  add  to  the  weight  obtained  4.8  mg.  for  each  100  cc.  of  filtrate  and 
washings. 

If  the  urine  under  examination  contains  albumin,  the  latter  must  be  coagu- 
lated by  heating  with  a  drop  or  two  of  acetic  acid  and  filtered  out,  before  the  test 
is  made.  If  the  urine  is  very  cold  to  begin  with  and  has  a  sediment  of  urates,  the 
latter  must  be  brought  into  solution  by  warming  before  beginning  the  test.  To 
prevent  precipitation  of  phosphates  during  the  warming  a  few  drops  of  hydro- 
chloric acid  may  be  added.     This  method  gives  only  approximate  results. 

Ammonium  Sulphate  Precipitation.  It  has  been  found  that  the  addition  of 
certain  ammonium  salts  to  urine  produces  a  precipitate  of  ammonium  urate  which 
is  practically  complete.  Fokker  and  Hopkins  recommended  the  chloride.  Folin  sug- 
gested the  sulphate,  which  possesses  some  advantages,  as  follows : 

A  special  precipitating  mixture  is  employed  which  in  1000  cc.  contains  500  gm. 
of  ammonium  sulphate,  5  gm.  of  uranium  acetate,  6  cc.  of  absolute  acetic  acid  and 
water  to  make  1  liter.  Measure  out  300  cc.  of  the  urine  and  add  75  cc.  of  the  above 
reagent.  Mix  well  and  allow  to  stand  five  minutes.  A  precipitate  containing  phos- 
phate and  some  protein  substance,  which  would  interfere  with  subsequent  work  if 
left,  separates.  Filter  off  two  portions  of  125  cc.  each,  equivalent  to  100  cc.  of  the 
original  urine,  and  to  each  add  5  cc.  of  strong  ammonia.  Allow  the  mixtures  to 
stand  24  hours,  in  which  time  a  complete  precipitation  of  ammonium  urate  takes 
place.  Collect  the  precipitates  on  a  small  filter  and  wash  practically  free  from 
chlorine  by  aid  of  10  per  cent,  ammonium  sulphate  solution.  Next  dissolve  the 
precipitates  in  100  cc.  of  hot  distilled  water,  add  15  cc.  of  pure  strong  sulphuric 
acid,  and  while  still  hot  titrate  the  mixture  with  N/20  potassium  permanganate 
solution,  each  cubic  centimeter  of  which  oxidizes  0.00375  gm-  of  uric  acid.  A  re- 
duction of  the  reagent,  with  loss  of  color,  follows.  When  the  uric  acid  is  fully 
oxidized  a  further  addition  of  permanganate  leaves  a  pink  tinge  in  the  liquid.  The 
addition  of  the  standard  reagent  from  the  burette  should  cease  as  soon  as  a  pink 
tinge  is  reached,  which  is  permanent  two  seconds  after  good  shaking.  By  waiting  a 
longer  interval  the  color  fades  and  more  solution  must  be  added  from  the  burette. 
If  the  reaction  is  stopped  with  the  first  decided  tinge  obtained,  as  explained,  for 
each  cubic  centimeter  of  permanganate  used  from  the  burette,  3.75  milligrams  of 
uric  acid  may  be  calculated  as  present.  In  illustration,  suppose  we  start  with  300 
cc.  of  urine  and  precipitate,  wash  and  dissolve  as  described.     If  now  we  run  12.5  cc. 


SOME    PRACTICAL    URINE    TESTS.  333 

of  the  twentieth  normal  permanganate  solution  into  the  hot  uric  acid  solution  to 
obtain  the  pink  color,  the  amount  of  this  acid  present  is  12.5  X  3-75  —  46.87  milli- 
grams in  the  100  cc.  As  ammonium  urate  is  slightly  soluble  in  the  mother  liquor, 
a  small  correction  must  be  added.  This  amounts  to  3  milligrams  for  each  100 
cubic  centimeters  of  the  urine  carried  through  to  the  titration.  Urine  contains 
traces  of  other  bodies  which  are  precipitated  with  the  uric  acid,  but  in  amount  so 
small  that  their  effect  may  be  practically  neglected.  The  duplicates  should  agree 
closely. 

THE    PURINE    BODIES. 

These  compounds  were  discussed  in  the  last  chapter.  Leaving  uric  acid  out 
of  consideration  the  amount  present  in  urine  is  not  large,  but  because  of  their 
relations  to  certain  diets  a  determination  is  often  a  matter  of  importance.  It  is 
not  practically  possible  to  make  an  accurate  separation  of  the  individual  purines, 
and  this  is,  besides,  not  necessary.  Several  methods  have  been  suggested  for  the 
group  precipitation,  and  of  these  the  following  gives  good  results  and  is  generally 
employed. 

This  method  depends  on  the  precipitation  of  the  purine  bodies  as  cuprous 
salts,  and  may  be  carried  out  in  this  way :  measure  out  200  cc.  of  urine  into  a  large 
casserole,  slightly  acidify  with  acetic  acid,  add  10  grams  of  sodium  acetate  and 
boil  about  a  minute.  Add  50  cc.  of  saturated  sodium  bisulphite  solution,  40  cc.  of 
10  per  cent  copper  sulphate  solution,  boil  again  and  filter.  Wash  the  precipitate 
with  hot  water  on  the  filter.  Return  the  precipitate  to  the  same  casserole  by  aid 
of  a  stream  of  hot  water,  add  25  cc.  of  strong  solution  of  sodium  sulphide,  and  then 
enough  acetic  acid  to  give  a  distinct  acid  reaction.  Boil  five  or  six  minutes  to 
expel  the  liberated  hydrogen  sulphide,  filter  and  wash  the  precipitated  copper 
sulphide  with  plenty  of  hot  water.  Save  the  filtrate  and  washings  in  the  same  casse- 
role in  which  the  process  was  begun.  This  solution  contains  an  approximately 
pure  mixture  of  the  purines,  free  from  other  nitrogenous  bodies  of  the 
original  urine.  By  repeating  the  precipitation  the  traces  of  foreign  substances 
may  be  removed.  Therefore  add  to  the  liquid  in  the  casserole  copper  sulphate  and 
the  bisulphite  solutions  as  before,  boil  several  minutes  and  filter.  Wash  the1  copper 
salts  thoroughly  with  hot  water,  and  finally  work  down  into  the  bottom  of  the 
filter,  which  should  not  be  large.  Allow  the  precipitate  to  drain  thoroughly,  and 
throw  it,  with  the  filter  paper,  into  a  Kjeldahl  flask  for  determination  of  nitrogen 
by  the  process  already  given.  It  may  be  well  to  add  35  to  40  cc.  of  strong  sul- 
phuric acid,  15  gm.  of  potassium  sulphate,  about  half  a  gram  of  copper  sulphate 
and  some  small  flakes  of  feather  tin  to  aid  in  the  oxidation,  which  under  these 
conditions  follows  easily.  A  clear  solution  is  obtained  in  less  than  one  hour.  This 
is  neutralized  with  pure  alkali  and  distilled  as  before  into  50  cc.  of  N/4  sulphuric 
acid. 

In  the  process  as  outlined  the  uric  acid  is  precipitated  with  the  other  purines, 
and  the  nitrogen  determined  is  the  nitrogen  of  all  the  purines,  including  the  uric 
acid.  Therefore,  the  uric  acid  nitrogen,  as  determined  above,  must  be  subtracted 
from  this  result  to  find  the  true  purine  nitrogen.  It  is  absolutely  essential  to 
employ  a  fresh  acid  sulphite  solution  in  the  original  precipitation  process  in  order 
to  secure  a  proper  reduction  and  precipitation  of  the  cuprous  salts.  The  acid  sul- 
phite solution  may  be  made  by  leading  a  good  current  of  sulphurous  oxide  (from 
copper  and  sulphuric  acid)  into  sodium  hydroxide  solution  until  saturation  is 
reached. 

CREATININE. 

This  product,  having  the  formula  GH7N.O,  occurs  normally  in  urine  and  is 
excreted  to  the  amount  of  1.5  to  2  grams  daily.  It  is,  therefore,  more  abundant 
than  uric  acid,  as  already  pointed  out.     As  it  is  readily  soluble  in  water  and  acids 


334  PHYSIOLOGICAL    CHEMISTRY. 

it  escapes  detection,  except  when  looked  for  by  special  reagents.  In  weak  solu- 
tions it  is  precipitated  by  phosphotungstic  acid,  phosphomolybdic  acid,  and  especially 
by  solutions  of  several  heavy  metallic  salts.  The  precipitate  given  with  a  neutral 
solution  of  zinc  chloride  is  the  most  characteristic.  It  gives  certain  color  reactions 
also.  The  following  test  may  be  applied  to  urine.  If  acetone  is  present  it  must  be 
expelled  by  heat.  To  about  25  cc.  of  this  urine  add  half  a  cubic  centimeter  of  a 
dilute  solution  of  sodium  nitroprusside  made  alkaline  with  caustic  soda.  With 
this  the  urine  gives  a  ruby-red  color,  fading  to  yellow.  Then  add  acetic  acid  in 
slight  excess  and  warm.    A  green  color  soon  appears,  deepening  finally  to  blue. 

With  picric  acid  creatinine  gives  a  very  characteristic  reaction,  suggested  by 
Jafre.  Add  to  the  urine  an  aqueous  solution  of  picric  acid  and  a  few  cc.  of  dilute 
sodium  hydroxide  solution.  A  deep  red  color  is  formed  almost  immediately,  which 
persists  a  long  time.  The  reaction  is  characteristic,  as  other  bodies  in  the  urine 
do  not  give  it.  Folin  has  made  the  reaction  the  basis  of  a  quantitative  colorimetric 
test  as  follows : 

Determination  of  Creatinine.  This  is  accomplished  by  aid  of  a  colorimeter 
in  which  the  color  of  the  above  Jafre  reaction  is  compared  with  the  color  from  a 
known  creatinine  solution,  or  from  a  standard  dichromate  solution.  Folin  suggests 
the  Duboscq  colorimeter  for  the  purpose,  but  other  forms  may  be  used,  and  employs 
a  half-normal  potassium  dichromate  solution,  with  24.54  grams  to  the  liter  as  the 
color  standard. 

Measure  10  cc.  of  urine  into  a  500  cc.  flask,  add  15  cc.  of  saturated  picric  acid 
solution  and  5  cc.  of  10  per  cent,  sodium  hydroxide  solution.  Allow  the  mixture 
to  stand  5  or  6  minutes  and  dilute  to  the  mark  with  distilled  water.  Meanwhile 
pour  dichromate  solution  into  one  of  the  cylinders  of  the  colorimeter  and  adjust 
to  a  depth  of  exactly  8  millimeters.  Pour  some  of  the  urine  solution  into  the 
other  cylinder  and  adjust  the  depth  until  the  shades  are  the  same,  leaving  the  dichro- 
mate side  at  8  millimeters.  An  exact  reading  of  the  depth  of  the  urine-picric  acid 
layer  is  noted,  and  a  calculation  of  the  strength  is  made  from  this  basis :  Folin  found 
that  10  mg.  of  pure  creatinine  in  500  cc,  under  the  same  conditions,  gave  such  a 
color  that  a  layer  of  8.1  mm.  was  equivalent  to  the  8  mm.  of  the  dichromate.  The 
concentration  of  the  solution  is  inversely  proportional  to  the  depth  of  layer  re- 
quired to  match  the  constant  standard.  Suppose  this  is  a  millimeters.  Then 
10  X  (8.1/a)  =  x.  If,  for  example,  we  read  off  a  depth  of  7.5  mm.,  x=  10.8  mg. 
of  creatinine  in  the  dilute  solution.  If  the  calculation  shows  over  15  mg.  or  below  5 
mg.  it  is  necessary  to  make  a  new  dilution  with  a  smaller  or  larger  volume  of  urine, 
to  get  the  most  accurate  results,  as  the  comparison  is  based  on  a  mean  content  of 
10  mg.  of  creatinine  to  500  cc.  after  the  dilution.  The  colorimetric  reading  should 
always  be  made  without  delay. 

Creatine  may  be  determined  in  the  same  manner  after  conversion  into  crea- 
tinine by  prolonged  boiling  with  dilute  hydrochloric  acid,  and  subsequent  neutra- 
lization. 

HIPPURIC   ACID. 

The  amount  of  this  acid  in  the  urine  is  not  large,  ordinarily,  but  may  be 
increased  by  the  consumption  of  certain  fruits  and  vegetables,  especially  by  those 
containing  benzoic  acid.  The  methods  of  determination  proposed  are  not  very 
exact.     The  following  is  the  best  one. 

Measure  out  200  to  300  cc.  of  urine,  make  it  alkaline  with  sodium  carbonate 
and  filter.  Evaporate  the  filtrate  nearly  to  dryness  and  extract  it  four  or  five  times 
by  shaking  with  alcohol.  Unite  the  alcoholic  extracts  and  distill  off  all  the  alcohol. 
The  aqueous  residue  is  acidified  with  hydrochloric  acid  and  extracted  by  shaking 
with  five  or  six  portions  of  pure  acetic  ether.  The  hippuric  acid  is  dissolved  in 
this  way,  and  the  united  extracts  are  partially  purified  by  washing  with  a  little 


SOME    PRACTICAL    URINE    TESTS.  335 

water.  The  acetic  ether  is  evaporated  to  dryness  at  a  moderate  temperature.  A 
residue  of  hippuric  acid  with  traces  of  other  substances  is  left.  Most  of  these 
impurities  may  be  removed  by  washing  this  residue  with  light  petroleum  ether,  in 
which  fats,  benzoic  acid  and  certain  other  things  are  soluble. 

The  hippuric  acid  left  after  this  treatment  may  be  still  further  purified  by 
dissolving  it  in  a  little  hot  water,  heating  with  well-burned  animal  charcoal,  filtering 
hot  and  evaporating  the  colorless  filtrate  slowly  to  dryness.  A  crystalline  residue 
should  now  be  obtained.  When  properly  carried  out  90  per  cent  of  the  hippuric 
acid  in  the  urine  may  be  removed  by  this  general  method. 

THE  PHOSPHATES  IN  URINE. 

Phosphoric  acid  occurs  normally  in  the  urine  combined  with  alkali  and  alkali- 
earth  metals,  of  which  combinations  the  alkali  phosphates  are  soluble  in  water, 
while  the  earthy  phosphates  are  insoluble.  In  the  urine,  however,  they  are  held 
in  solution  through  several  agencies.  The  larger  part  of  the  earthy  phosphates 
appear  to  be  held  here  normally  in  the  acid  condition;  that  is,  as  compounds  of 
the  formulas  CaH^PCX)*  and  MgH«(PO*)a.  The  salts  of  the  type  CaHPO*  are 
present,  also,  in  small  amount.  As  long  as  the  urine  maintains  its  acid  reaction 
these  bodies  may  be  expected  to  remain  in  solution,  but  if  it  becomes  alkaline  by 
fermentation,  or  by  the  addition  of  the  hydroxides  or  carbonates  of  ammonium, 
sodium,  or  potassium,  the  acid  phosphates  are  converted  into  insoluble,  neutral 
phosphates  and  precipitated.  Most  urines  contain  along  with  the  acid  phosphates 
'traces  of  neutral  phosphates  which  precipitate  on  boiling.  It  has  been  suggested 
that  these  phosphates  are  held  by  traces  of  ammonium  compounds  or  by  carbonic 
acid,  both  of  which  are  driven  off  by  heat,  allowing  the  phosphates  to  precipitate. 
It  is  well  known,  however,  that  some  urines  can  be  boiled  without  showing  any 
sign  of  precipitation.  In  such  cases  it  is  probable  that  the  neutral  phosphates  are 
not  present. 

Part  of  the  phosphoric  acid  of  the  urine  comes  directly  from  the  phosphates 
of  the  food  and  another  portion  results  from  the  oxidation  of  the  phosphorus- 
holding  tissues.  In  health,  the  rate  of  such  oxidation  is  practically  constant,  or 
nearly  so,  but  in  disease  it  may  be  greatly  increased  or  diminished.  Variations  in 
the  amount  of  excreted  phosphates  may  therefore  become  of  considerable  clinical 
importance. 

Various  statements  are  found  in  the  books  regarding  the  mean  excretion  of 
the  alkali  and  earthy  phosphates.  Different  observers  have  reported  between  2  and 
5  grams  of  phosphoric  anhydride  (P2O0),  while  3  grams  may  be  taken,  perhaps  as 
the  mean. 

The  recognition  of  the  phosphates  is  an  extremely  easy  matter.  The  presence 
of  earthy  phosphates  may  be  shown  by  adding  to  the  urine  enough  ammonia  water 
to  give  a  faint  alkaline  reaction  and  then  warming.  A  flocculent  precipitate,  re- 
sembling albumin,  appears  and  is  usually  white,  or  nearly  so.  But  sometimes  color- 
ing-matters come  down  with  it  in  amount  sufficient  to  give  it  a  brownish  or  reddish 
shade.  It  will  be  recalled  that  the  color  of  this  precipitate  was  referred  to  under 
the  head  of  blood  tests. 

The  alkali  phosphates  can  be  detected  in  the  filtrate  after  separation  of  the 
earthy  phosphates.  To  this  end,  add  to  the  clear  alkaline  liquid  a  little  more  am- 
monia and  some  clear  magnesia  mixture.  A  fine  crystalline  precipitate  of  am- 
monium-magnesium phosphate  separates  and  settles  rapidly.  This  is  very  char- 
acteristic. The  qualitative  tests  for  phosphates  have,  however,  little  value  in  ex- 
amination of  the  urine.  We  are  chiefly  concerned  with  the  amount,  the  measure- 
ment of  which  will  now  be  described. 

Determination    of   Phosphates.     It    is    customary    to    measure   the    total    phos- 


336  PHYSIOLOGICAL    CHEMISTRY. 

phoric  acid,  not  the  alkali  or  earthy  phosphates,  separately.  We  have  at  our  dis- 
posal several  methods,  gravimetric  and  volumetric,  of  which  the  latter  are  accurate 
and  most  convenient.  A  volumetric  process  will  be  described  which  serves  for  the 
measurement  of  the  phosphoric  acid  as  a  whole,  and  which  can  be  used  for  the 
separate  measurement  of  the  earthy  and  alkali  phosphates  by  dealing  with  the  pre- 
cipitate and  filtrate  described  in  the  qualitative  test  above.  This  method  depends 
on  the  fact  that  solutions  of  uranium  nitrate  or  acetate  precipitate  phosphates  in 
greenish-yellow  colored,  flocculent  form,  and  that  in  a  solution  holding  in  sus- 
pension a  precipitate  of  uranium  phosphate  any  excess  of  soluble  uranium  com- 
pound may  be  recognized  by  the  reddish  brown  precipitate  which  it  gives  with  a 
solution  of  potassium  ferrocyanide.  The  latter  substance  serves,  therefore,  as 
an  indicator.  If  to  a  phosphate  solution  in  a  beaker  a  dilute  uranium  solution  be 
added  precipitation  continues  until  the  whole  of  the  phosphates  have  gone  into 
combination  with  the  uranium.  If,  during  the  precipitation,  drops  of  liquid  from 
the  beaker  are  brought  in  contact  with  drops  of  fresh  ferrocyanide  solution  on  a 
glass  plate,  no  reddish  brown  precipitate  of  uranium  ferrocyanide  appears  until  the 
last  trace  of  uranium  phosphate  has  been  formed.  The  production  of  uranium 
ferrocyanide  is  the  indication,  therefore,  of  the  finished  precipitation  of  the 
phosphate. 
The  reaction  between  uranium  and  phosphates  is  shown  by  the  equation : 

U02(N03)2  +  KH2P04  =  U02HP04  +  KN03  +  HN03 

From  this  it  follows  that  238.5  parts  of  uranium  are  required  for  71  parts  of* 
P205.  In  order  to  have  the  reaction  take  place  as  above  it  is  necessary  to  neutralize 
the  nitric  acid  as  fast  as  formed,  or  dispose  of  it  in  some  other  manner.  The  best 
plan  is  to  add  to  the  solution  some  sodium  acetate  and  acetic  acid.  The  latter 
brings  the  phosphates  into  the  form  of  acid  salts  while  the  acetate  decomposes 
with  formation  of  sodium  nitrate  and  free  acetic  acid,  which  does  not  interfere 
with  the  reaction. 

We  need  the  following  reagents : 

(a)  Standard  Uranium  Solution.  This  is  made  by  dissolving  36  gm.  of  the 
pure  crystallized  nitrate,  U02(N03)2-6H20,  in  water  to  make  one  liter. 
The  solution  is  standardized  as  below : 

(b)  Standard  Phosphate  Solution.  This  is  made  by  dissolving  10.087  gm.  of 
pure  crystals  of  sodium  phosphate,  HNa2P04i2H20,  to  make  one  liter. 
50  cc.  of  the  solution  contains  100  mg.  of  P205. 

(c)  Sodium  Acetate  Solution.  Dissolve  100  grams  in  800  cc.  of  distilled 
water,  add  100  cc.  of  30  per  cent,  acetic  acid  and  then  water  enough  to 
make  one  liter. 

(d)  Fresh  Ferrocyanide  Solution.  Dissolve  10  grams  of  pure  potassium 
ferrocyanide  in  100  cc.  of  distilled  water.  The  solution  should  be  kept  in 
the  dark. 

The  actual  value  of  the  uranium  solution  is  determined  by  the  following  ex- 
periment. Measure  out  50  cc.  of  the  phosphate  solution,  (&),  add  5  cc.  of  the  acet- 
ate solution,  (c),  and  heat  in  a  beaker  in  a  water-bath  to  near  the  boiling  temper- 
ature. Place  several  drops  of  the  ferrocyanide  solution  on  a  white  plate.  Fill  a 
burette  with  the  uranium  solution  and  when  the  solution  in  the  beaker  has  reached 
the  proper  temperature  run  into  it  from  the  burette  18  cc.  of  the  uranium  standard. 
Warm  again,  and  by  means  of  a  glass  rod  bring  a  drop  of  the  liquid  in  the  beaker 
in  contact  with  one  of  the  ferrocyanide  drops  on  the  plate.  If  the  uranium  solu- 
tion has  been  properly  made  no  red  color  should  yet  appear.  Now  run  in  a  fifth  of 
1  cc.  more  from  the  burette,  warm  and  test  again,  and  repeat  these  operations  until 


SOME    PRACTICAL    URINE    TESTS.  337 

the  first  faint  reddish  shade  begins  to  show  on  bringing  the  two  drops  in  contact. 
With  this  test  as  a  preliminary  one  make  a  second,  adding  at  first  one-fifth  of  a 
cubic  centimeter  less  than  the  final  result  of  the  preliminary,  and  finish  as  before. 
Something  less  than  20  cc.  should  be  needed  to  complete  the  reaction.  Supposing 
19.8  cc.  are  required  for  the  purpose,  the  whole  solution  should  be  diluted  in  the 
proportion, 

19.8   :  20   :    :  a    :  x 

in  which  a  represents  the  volume  on  hand.  Each  cubic  centimeter  precipitates 
exactly  5  mg.  of  P205.  The  liter  contains  35.38  gm.  of  the  true  uranium  nitrate, 
which,  if  the  salt  were  absolutely  pure,  could  be  weighed  out  directly. 

The  test  of  the  urine  is  made  exactly  as  above.  Measure  out  50  cc,  add  5  cc. 
of  the  acetate  mixture  and  finish  as  before.  The  50  cc.  of  urine,  in  the  mean 
contains  about  as  much  phosphoric  acid  as  was  present  in  the  same  volume  of 
standard  phosphate  solution.  The  titration  must  be  made  hot,  because  the  reaction 
is  much  quicker  and  sharper  in  hot  solution  than  in  cold.  Make  always  two  tests; 
the  first  is  an  approximation,  while  the  second  gives  a  much  closer  result. 

A  separate  test  of  the  earthy  phosphates  may  be  made  by  adding  to  200  cc.  of 
urine  enough  ammonia  to  give  an  alkaline  reaction.  The  urine  then  must  stand 
until  the  precipitated  phosphates  settle  out.  The  precipitate  is  collected  on  a 
small  filter,  washed  with  water  containing  a  very  little  ammonia,  and  then  allowed 
to  drain.  It  is  next  dissolved  in  a  small  amount  of  acetic  acid,  the  solution 
diluted  to  50  cc,  mixed  with  5  cc.  of  the  sodium  acetate  solution  and  titrated  as 
before.  The  reaction  here  is  not  quite  as  accurate  as  with  the  alkali  phosphate, 
but  the  results  are  satisfactory  for  the  purpose.  The  difference  between  the  total 
phosphates  and  the  earthy  phosphates,  expressed  in  terms  of  P205,  is  the  amount 
combined  as  alkali  phosphates. 

It  is  also  possible  to  determine  the  amount  of  phosphoric  acid  combined  as 
monohydrogen  salt  and  that  combined  as  dihydrogen  salt,  but  the  determination 
has  at  the  present  time  little  clinical  value. 

Instead  of  finding  the  end  point  in  the  precipitation  with  uranium  solution  by 
means  of  drops  of  ferrocyanide  as  explained,  the  following  process  may  be  fol- 
lowed. Add  to  the  urine  the  sodium  acetate  as  before  and  then  three  or  four 
drops  of  tincture  of  cochineal.  Heat  to  boiling  and  add  the  uranium  solution  to 
the  hot  liquid.  Just  as  soon  as  the  phosphate  is  combined  and  a  trace  of  uranium 
left  in  excess  it  produces  a  green  color  or  precipitate  with  the  cochineal,  which 
thus  serves  as  an  indicator  to  show  the  end  of  the  reaction.  If  the  urine  is  quite 
warm  the  color  is  sharp. 

THE  CHLORIDES  IN  URINE. 

Practically  all  the  chlorine  consumed  with  the  food,  mainly  in  common  salt, 
is  eliminated  in  the  urine.  The  excretion,  therefore,  varies  within  wide  limits  in 
different  individuals,  but  in  the  mean  amounts  to  10  or  15  gm.  daily  of  the  salt. 
A  large  increase  in  excreted  chlorine  points  merely  to  increased  consumption,  but 
a  marked  decrease  may  point  to  one  of  several  pathological  conditions.  Quantita- 
tive tests  have  sometimes  considerable  value,  and  are  easily  made  through  the 
reaction  between  silver  nitrate  and  a  chloride,  by  a  volumetric  process. 

Determination  of  Chlorine.  The  reaction  between  nitrate  and  chloride  is 
expressed  as  follows : 

NaCl  -J-  AgNO,  =  AgCl  +  NaNO, 

from  which  it  appears  that  5.85  mg.  of  sodium  chloride  require  for  precipitation 
16.997  rng-  of  silver  nitrate.     A  standard  silver  solution  may  be  made  then  of  this 

23 


338  PHYSIOLOGICAL    CHEMISTRY. 

strength,  by  dissolving  16.997  gm-  of  the  pure  fused  nitrate  to  make  1  liter  with 
distilled  water. 

In  one  method  of  determination  10  cc.  of  urine  is  evaporated  in  a  platinum 
or  porcelain  dish  to  dryness,  after  mixing  with  2  gm.  of  potassium  nitrate,  and 
1  gm.  of  sodium  carbonate.  The  dry  residue  is  carefully  fused,  and  the  resulting 
mass  dissolved  in  water.  After  exactly  neutralizing  with  nitric  acid,  and  adding 
a  little  potassium  chromate  as  indicator  the  chlorine  is  titrated  in  the  usual  manner. 
But  the  process  is  not  generally  followed,  being  replaced  by  the  next  one. 

Volhard's  Method.  We  have  here  a  method  by  which  the  chlorine  in  urine 
can  be  quickly  and  accurately  determined  without  fusion.  The  principle  involved 
in  the  process  is  this.  If  to  a  chloride  solution  a  definite  volume  of  standard  silver 
solution  be  added,  and  this  in  excess  of  that  necessary  to  precipitate  the  chloride, 
the  amount  of  this  excess  can  be  found  by  another  reaction,  subtracted  and  leave 
as  the  difference  the  volume  actually  needed  for  the  chloride.  The  reaction  for 
the  excess  depends  on  these  facts.  A  thiocyanate  solution  gives  with  silver  nitrate 
solution  a  white  precipitate  of  silver  thiocyanate,  AgSCN.  It  also  gives  with  a 
ferric  solution  a  deep  red  color  due  to  the  formation  of  soluble  ferric  thiocyanate, 
FeS3(CN)3.  If  the  silver  and  ferric  solutions  are  mixed  and  the  thiocyanate  added 
the  second  reaction  does  not  begin  until  the  first  is  completed;  that  is,  the  silver 
must  be  first  thrown  down  as  white  thiocyanate  before  a  permanent  red  shade  of 
ferric  thiocyanate  appears.  The  presence  of  silver  chloride  interferes  but  slightly 
with  these  reactions.  Therefore,  if  we  have  a  thiocyanate  solution  of  definite 
strength  we  can  use  it  with  the  ferric  indicator  to  measure  the  excess  of  silver 
used  after  precipitating  the  chlorine  solution. 

The  reaction  between  silver  nitrate  and  a  thiocyanate  is  expressed  by  the  follow- 
ing equation : 

AgN03  +  NH4SCN  =  AgSCN  +  NH4N03. 

For  16.99  milligrams  of  the  silver  nitrate  we  use  7.6  milligrams  of  the  thiocyanate. 
In  this  method  the  standard  solutions  required  are 

(a)  Standard  Silver  Nitrate  Solution,  N/10. — Made  as  before  with  16.997 
grams  of  the  fused  salt  to  the  liter. 

(b)  Standard  Thiocyanate  Solution.  Weigh  out  about  7.7  gm.  of  ammo- 
nium thiocyanate  and  dissolve  to  make  1  liter.  Adjust  the  exact  strength 
as  below: 

(c)  Ferric  Indicator.  Use  for  this  a  strong  solution  of  ferric  alum,  free 
from  chlorine. 

To  find  the  exact  strength  of  the  thiocyanate  solution  proceed  as  follows : 
Measure  into  a  flask  or  beaker  25  cc.  of  the  N/10  silver  nitrate,  and  add  about 
3  cc.  of  the  strong  ferric  alum  solution.  Add  also  enough  strong,  pure  nitric  acid 
to  make  a  perfectly  clear  mixture,  for  which  not  over  2  or  3  cc.  should  be  needed. 
From  a  burette  run  in  the  thiocyanate  solution  a  little  at  a  time,  shaking  after  each 
addition.  A  red  color  appears  temporarily,  but  vanishes  on  shaking.  After  a 
time  this  color  disappears  very  slowly,  which  shows  that  the  end  point  is  near. 
The  burette  solution  is  therefore  added  more  carefully,  best  by  drops,  until  at 
last  a  single  drop  is  sufficient  to  give  a  permanent  reddish  tinge.  Something  less 
than  25  cc.  should  be  used  for  this.  Repeat  the  test  and  if  the  same  result  is  found 
dilute  the  thiocyanate  solution  so  as  to  make  25  cc.  of  the  volume  used  in  the  titra- 
tion. For  instance,  if  24.2  cc.  were  required  900  cc.  of  the  solution  may  be  diluted 
in  this  proportion : 

24.2   :  25   ::  900   :  x  .'.x  =  929.8. 

We   have   now   a   standard   thiocyanate   solution    corresponding    exactly   to    the 


SOME    PRACTICAL    URINE    TESTS.  339 

silver  solution.  To  test  it  further  and  illustrate  its  use  with  chlorides  measure 
out  25  cc.  of  an  N/10  sodium  chloride  solution,  very  accurately  prepared,  and  add 
to  it,  from  a  burette,  exactly  30  cc.  of  the  silver  nitrate  solution,  then  the  ferric 
indicator  and  the  nitric  acid  as  given  above.  Shake  the  mixture  and  filter  it 
through  a  small  filter  into  a  clean  flask  or  beaker.  Wash  out  the  vessel  in  which 
the  precipitate  was  made  with  about  20  cc.  of  pure  water,  pouring  the  washings 
through  the  filter.  Then  wash  the  filter  with  about  20  cc.  more  of  water,  allowing 
the  washings  to  mix  with  the  first  filtrate.  This  mixed  filtrate  contains  all  the 
silver  used  in  excess  of  the  chloride.  Now  bring  it  under  the  thiocyanate  burette 
and  add  this  solution  until  a  reddish  tinge  becomes  permanent.  Exactly  5  cc. 
should  be  necessary  for  this. 

The  chlorides  of  the  urine  may  be  treated  in  about  the  same  manner.  To  a 
measured  volume  of  the  urine,  usually  10  cc,  an  excess  of  silver  nitrate  solution 
is  added ;  25  cc.  with  most  urines  is  enough,  and  then  the  indicator  and  acid. 

But  as  the  coloring  matters  in  urine  interfere  somewhat  with  the  sharpness 
of  the  titration  it  is  best  to  destroy  them  by  partial  oxidation  with  sodium  peroxide 
or  potassium  permanganate.  The  latter  is  preferable.  To  10  cc.  of  urine  add  3  cc. 
of  pure,  strong  nitric  acid,  then  the  ferric  alum.  Add  also  3  or  4  drops  of  a 
saturated,  chlorine-free,  solution  of  potassium  permanganate.  The  red  color  which 
forms  at  first,  soon  disappears.  Then  add  25  cc.  of  the  N/10  silver  nitrate,  stir 
up,  filter  and  titrate  the  excess  of  silver  with  thiocyanate,  as  above.  If  the  first 
drops  run  in  from  the  thiocyanate  burette  produce  a  red  color  it  is  evidence  that 
there  is  no  silver  in  excess,  and  that  the  urine  was  very  strong  in  chloride.  In 
this  case  start  a  new  test  with  urine  diluted  one-half. 

To  illustrate  the  calculation,  if  we  use  10  cc.  of  urine,  25  cc.  of  silver  nitrate 
and  finally  3.4  cc.  of  thiocyanate,  25 — 3.4  =  21.6  cc,  the  amount  of  silver  nitrate 
actually  needed  for  the  chloride.  Then,  21.6  X  3-55 =  76.68  mg.,  the  amount  of 
chlorine  in  the  urine.  This  is  equivalent  to  126.36  mg.  of  sodium  chloride,  or 
12.636  gm.  per  liter. 

THE  TOTAL   SULPHUR  AND   SULPHATES   IN  URINE. 

It  was  shown  in  the  last  chapter  that  sulphur  appears  in  the  urine  in  the 
ordinary  mineral  sulphates,  in  the  organic  or  ethereal  sulphates  and  in  the  so-called 
neutral  or  unoxidized  form.  The  total  sulphur  is  determined  by  oxidizing  every- 
thing to  the  condition  of  mineral  sulphate,  and  precipitating  as  barium  sulphate. 
As  oxidizing  agents  which  may  be  used  with  urine  the  following  have  been  tried : 
fuming  nitric  acid,  sodium  peroxide,  potassium  or  other  nitrate  and  chlorates. 
The  method  given  below,  which  was  worked  out  by  Benedict  in  the  author's 
laboratory  yields  good  results. 

Total  Sulphur.  An  oxidizing  reagent  is  made  by  dissolving  200  grams  of 
pure  copper  nitrate  and  50  grams  of  potassium  chlorate  in  water  to  make  1  liter. 
To  10  cc.  of  urine  in  a  small  porcelain  dish  add  5  cc.  of  the  above  reagent  and 
evaporate  to  dryness  over  a  low  flame,  then  increase  the  heat  gradually  and  bring 
up  to  a  high  temperature  with  a  good  Bunsen  flame.  Continue  the  heat  five  to  ten 
minutes  after  the  mass  has  fused  and  solidified.  Finally  allow  the  dish  to  cool, 
add  10  cc.  of  10  per  cent  hydrochloric  acid,  and  warm  until  solution  takes  place. 
Filter  into  a  small  Erlenmeyer  flask,  wash  the  filter  thoroughly  and  make  the 
filtrate  up  to  150  cc.  Add  now,  slowly,  10  cc.  of  5  per  cent  barium  chloride 
solution,  best  drop  by  drop,  -lir  gently  and  allow  to  stand  an  hour.  At  the  end  of 
this  time  collect  the  precipitate  on  a  weighed  Gooch  crucible  in  the  usual  manner. 
The  increase  in  vreighl  gives  the  barium  sulphate  from  the  total  sulphur. 

Total  Sulphates.  The  method  recommended  by  Folin  gives  uniform  results. 
It  is  as  follows:   Measure  into  an    Erlenmeyer  flask  of  250  cc.  capacity,  25  cc.  of 


340  '  PHYSIOLOGICAL    CHEMISTRY. 

urine  and  20  cc.  of  8  per  cent  hydrochloric  acid.  Boil  gently  half  an  hour,  with 
a  watch  glass  or  small  beaker  over  the  neck  of  the  flask.  At  the  end  of  this  time 
cool  and  dilute  with  cold  water  to  150  cc.  Add  10  cc.  of  5  per  cent  barium  chloride 
solution,  without  agitating  more  than  is  necessary  to  mix  the  liquids.  If  the 
barium  chloride  is  added  very  slowly  from  a  dropping  tube  no  further  agitation 
is  necessary.  At  the  end  of  about  an  hour  filter  through  a  weighed  Gooch  crucible 
and  wash  with  250  cc.  of  cold  water.     Dry  and  weigh  as  usual. 

In  this  method  of  determination  the  long  boiling  of  the  urine  with  the  hydro- 
chloric acid  effects  the  splitting  of  the  ethereal  sulphates,  so  that  in  the  following 
barium  chloride  precipitation  they  come  down  with  the  inorganic  sulphates.  The 
latter  may  be  found  separately,  as  recommended  by  Folin. 

Inorganic  Sulphates.  Dilute  25  cc.  of  urine  with  10  cc.  of  8  per  cent  hydro- 
chloric acid  and  water  to  make  150  cc.  in  an  Erlenmeyer  flask  of  250  cc.  capacity. 
Add  slowly,  from  a  dropping  pipette,  10  cc.  of  5  per  cent  barium  chloride  solution, 
without  shaking.  Allow  the  mixture  to  stand  an  hour  and  then  filter  through  a 
weighed  Gooch  crucible.  Wash  the  precipitate  with  250  cc.  of  cold  water,  dry  and 
ignite  in  such  a  manner  that  the  barium  sulphate  is  protected  from  reducing  gases. 

By  the  conditions  of  the  precipitation  the  ethereal  sulphates  are  not  decom- 
posed, as  the  liquid  was  not  heated  during  the  operation.  From  the  weight  of  the 
barium  sulphate  the  sulphur  should  be  calculated  in  each  case  for  a  given  volume. 
The  difference  between  the  two  results  is  the  sulphur  in  the  form  of  ethereal 
sulphates. 

If  the  difference  is  taken  between  the  sulphur  of  the  "total  sulphur "  test  and 
the  "  total  sulphate "  test  the  result  is  the  "  neutral "  or  unoxidized  sulphur. 

THE   SEDIMENT   FROM   URINE. 

Urine  is  frequently  cloudy  when  passed  and  on  standing  deposits  a  sediment 
of  the  substances  imparting  the  cloudiness.  Other  urines  which  may  appear  per- 
fectly clear  at  first  also  throw  down  deposits  after  a  time.  This  is  always  the 
case  with  urine  allowed  to  stand  long  enough  to  undergo  alkaline  fermentation, 
when  a  precipitate  of  phosphates  forms.  The  deposit  is  frequently  caused  by  a 
change  of  temperature.  Warm  voided  urine  holding  an  excess  of  urates  may  be 
perfectly  clear,  but  becomes  cloudy  as  its  temperature  goes  down  with  the  forma- 
tion of  a  light  reddish  sediment.  This  is  a  perfectly  normal  action,  and  indeed 
most  sediments  may  be  considered  in  the  same  light.  Urine  containing  a  deposit 
is  not  necessarily  pathological. 

There  are  conditions,  however,  in  which  the  sediment  is  an  indication  of  ab- 
normality, and  its  examination  becomes  important  clinically.  Certain  sediments 
are  pathological  because  of  their  origin,  others  because  of  their  amount.  For  in- 
stance, blood  and  pus  corpuscles,  casts  of  the  uriniferous  tubules  of  the  kidney 
and  a  few  other  forms  are  not  found  normally  in  urine,  and  their  presence  is  of 
importance,  whether  observed  in  large  or  small  quantity.  Sediments  containing 
phosphates,  uric  acid  and  urates,  calcium  oxalate  and  other  salts,  are  common 
enough  and  usually  attract  no  attention,  but  if  the  amount  of  these  deposits  is 
very  large  there  may  be  attached  to  them  clinical  significance  and  they  deserve 
study. 

In  the  examination  of  a  sediment  it  is  necessary  to  allow  the  urine  to  stand 
long  enough  to  deposit  the  important  forms  it  may  contain,  which  may  require 
twenty-four  hours  or  more.  For  the  deposition  of  a  sediment  the  urine  should  be 
left  in  a  place  with  an  even  temperature,  preferably  not  above  150  C.  A  low 
temperature  favors  the  precipitation  of  urates,  while  decomposition  may  begin  if 
the  temperature  be  allowed  to  go  up.  Some  of  the  light  organic  forms  have  a 
specific  gravity  so  little  above  that  of  the  urine  that  they  may  remain  a  long  time 


SOME    PRACTICAL    URINE    TESTS.  341 

in  suspension.  It  is  important,  therefore,  to  allow  plenty  of  time  for  these  to 
settle.  If  the  weather  is  warm  and  there  is  no  good  means  at  hand  for  keeping 
the  temperature  of  the  urine  down  until  the  examination  can  be  made,  or  if  for 
any  reason  this  must  be  delayed  for  some  days,  it  is  well  to  add  some  preservative 
to  the  urine;  i.  e.,  something  to  prevent  fermentation.  Many  substances  have 
been  suggested  for  this  purpose,  some  of  which  are  very  objectionable  inasmuch 
as  they  form  precipitates  which  often  obscure  what  is  sought  for.  Chloroform  is 
the  simplest  and  at  the  same  time  one  of  the  best  substances  which  can  be  added. 

To  ioo  cc.  of  the  urine  to  be  set  aside  for  tests  add  three  or  four  drops  of 
chloroform  and  dissolve  by  shaking.  It  is  not  well  to  add  more  than  this,  as  there 
is  danger  of  leaving  minute  droplets  undissolved,  and  these  are  confusing  in  the 
subsequent  examination.  The  chloroform  may  be  applied  in  the  form  of  aqueous 
solution.  Add  about  10  grams  of  chloroform  to  a  liter  of  distilled  water  and  shake 
thoroughly;  about  three-fourths  will  dissolve  at  the  ordinary  temperature;  25 
cc.  of  this  saturated  solution  may  be  added  to  100  cc.  of  the  urine  to  be  examined, 
which  is  then  allowed  to  stand  as  before. 

Recently,  formaldehyde  has  come  into  use  as  a  urine  preservative  and  is  applied 
as  is  the  chloroform.  It  must  be  remembered  that  both  of  these  substances  are 
reducing  agents,  and  therefore  should  not  be  used  with  urine  to  be  tested  for  sugar. 

All  of  these  methods  of  preservation  are  unnecessary  if  a  centrifuge  is  at  hand, 
with  which  a  deposit  from  about  10  cc.  of  urine  may  be  secured  in  a  few  minutes. 
This  plan  is  always  preferable,  and  is  now  generally  followed. 

After  the  deposit  has  settled  pour  off  the  supernatant  liquid  very  carefully  and 
by  means  of  a  small  pipette  with  a  coarse  opening  transfer  one  or  two  drops 
to  a  perfectly  clean  glass  slide.  Clean  a  cover  glass  with  great  care  and  by  means 
of  small  brass  forceps  lower  it  on  the  drop  of  liquid  in  such  a  manner  as  to  ex- 
clude air  bubbles.  This  can  be  done  by  lowering  it  inclined  to  the  slide,  not 
parallel  with  it,  so  as  to  touch  the  liquid  on  one  side  first.  In  settling  down,  the 
cover  now  pushes  the  air  in  front  of  it  and  gives  a  field  generally  free  from  bub- 
bles. The  slide  is  then  examined  under  a  microscope  with  a  magnifying  power  of 
250  to  300  diameters.  Either  natural  or  artificial  light  may  be  used,  but  it  must  not 
be  very  bright.  A  very  common  mistake  in  the  examination  of  urinary  sediments 
by  the  microscope  is  to  employ  so  high  a  degree  of  illumination  that  the  lighter 
and  nearly  transparent  bodies  are  completely  overlooked. 

Sediments  from  urine  are  commonly  classed  as  organized  and  unorganized,  these 
divisions  being  then  subdivided  according  to  various  plans.  The  important  forms 
under  each  division  are  shown  in  the  following  schemes : 

Organized   Sediments.  Unorganized   Sediments. 

Blood  corpuscles.  Uric  acid. 

Mucus  and  pus  corpuscles.  Various  urates. 

Epithelium  from  various  locations.  Leucine  and  tyrosine. 

.Mucin  bands,  or  threads.  Cystin. 

Casts  of  the  uriniferous  tubules.  Cholesterol. 

Spermatozoa.  Fat  globules. 

Fungi.  Hippuric  acid. 

Certain  other  parasites.  Calcium  carbonate. 

Calcium  phosphate. 
Calcium  oxalate. 
Magnesium   phosphates. 
In   addition  to  these  there  are  often   found  in   the   urine  certain   bodies   whose 
presence  must  be  called  accidental;    for   instance,   hairs,   fibers   of  cotton,   silk   or 
wool,    starch    granules,    bits    of    wood,    mineral    dust,    etc.     Some   of    these    will   be 
referred  to  later. 


342  PHYSIOLOGICAL    CHEMISTRY. 

ORGANIZED  SEDIMENTS. 

Blood  Corpuscles.  Urine  containing  blood  presents  a  characteristic  appearance 
easily  recognized,  unless  it  be  present  in  very  small  quantity.  If  the  reaction  of 
the  urine  is  acid  the  color  is  generally  dark;  but  if  alkaline  the  shade  is  inclined 
to  reddish.  Blood  corpuscles  enter  the  urine  from  several  different  sources  and 
their  presence  is  usually  a  pathological  indication,  but  not  always,  as  they  may  come, 
for  instance,  from  menstruation.  The  kidneys,  or  their  pelves,  the  ureters,  the 
bladder,  the  urethra,  the  vagina,  or  the  uterus  may  be  the  seat  of  the  lesion  from 
which  the  blood  starts,  and  its  appearance  sometimes  gives  a  clue  to  its  origin. 

Fresh  blood  corpuscles  are  clear  in  outline  and  show  distinctly  their  bicon- 
cavity.  But  corpuscles  which  have  been  long  in  contact  with  the  urine  become 
much  swollen,  less  distinct  in  outline,  often  biconvex,  or  nearly  spherical  even, 
and  lighter  in  color.     As  long  as  the  reaction  of  the  urine  is  acid  the  corpuscles 

'•-'"->  ®  %®®o 

J®    *  °   o  0 

o°o    o 

Fig.  31,     Human  blood  corpuscles,  400  diameters. 

remain  comparatively  fresh  in  appearance,  but  with  the  beginning  of  the  alkaline 
reaction  disintegration  and  loss  of  color  soon  set  in. 

The  microscopic  recognition  of  blood  in  urine  is  easy  enough  if  it  is  not  too 
old.  The  fresh,  red  corpuscles  of  human  blood  have  a  mean  diameter  of  about 
0.0077  mm.,  but  when  swollen  by  absorption  of  water  they  are  somewhat  larger. 
When  seen  on  edge  they  appear  as  shown  at  the  left  in  the  figure  above.  If  pre- 
senting the  flat  side  to  the  eye,  they  appear  as  disks  whose  centers  grow  alternately 
light  and  dark  by  changing  the  focus  of  the  instrument.  In  old  urine,  especially 
with  alkaline  reaction,  they  appear  as  granulated  spheres,  shown  at  the  left  of 
the  figure.  In  all  cases  the  color  is  more  or  less  yellowish.  It  is  generally  assumed 
that  the  paler  washed-out  corpuscles  come  from  lesions  higher  up,  from  the  pelvis, 
or  kidney  even,  while  the  brighter  fresh  blood  suggests  a  lesion  nearer  the  point 
of  discharge;  that  is,  from  the  bladder  or  urethra.  This  is  pretty  certain  to  be 
the  case  if  the  blood  is  discharged  but  little  mixed  with  the  urine  and  settles 
rapidly  as  a  distinct  mass. 

The  crenated  appearance  of  the  corpucles,  shown  at  the  right  and  above  in  the 
figure,  is  due  to  contact  with  a  denser  medium,  to  partial  drying  and  sometimes 
to  certain  pathological  conditions,  while  the  swollen  appearance,  from  the  absorp- 
tion of  water,  is  illustrated  below. 

Mucus  and  Pus  Corpuscles.  These  are  white  corpuscles  somewhat  larger  than 
the  red  blood  corpuscles  and  spherical  in  outline.  The  term  leucocyte  is  frequently 
applied  to  these  as  well  as  to  the  so-called  white  corpuscles  of  blood.  Their  size 
varies  greatly  but  the  average  diameter  may  be  given  as  0.009  mm-  All  these 
corpuscles  present,  when  fresh,  a  slightly  granular  appearance  and  occasionally 
show  one  or  more  nuclei.     The  addition  of  a  little  acetic  acid  to  the  sediment 


SOME    PRACTICAL    URINE    TESTS.  343 

brings  the  nucleus  out  distinctly  so  that  it  may  be  seen  under  the  microscope  as  a 
characteristic  appearance. 

Mucus  corpuscles  in  small  number  are  normally  present  in  urine,  but  pus  cor- 
puscles enter  the  urine  as  a  constituent  of  pus  itself  which  is  an  albuminous  product 
discharged  from  suppurating  surfaces  and  not  normal.  It  has  been  pointed  out 
that  the  reactions  of  mucus  and  albumin  are  distinct,  but  urine  containing  pus 
always  affords  reactions  for  albumin.  Pus  in  urine  tends  to  form  a  sediment 
at  the  bottom  of  the  containing  vessel  and  may  be  recognized  by  the  following 
method  : 

Donne's  Test.  Pour  the  urine  from  the  sediment  and  add  to  the  latter  an 
equal  volume  of  thick  potassium  hydroxide  solution,  or  a  small  piece  of  the  solid 
potassa.  Stir  with  a  glass  rod.  The  strong  alkali  converts  the  pus  into  a  thick 
viscid  mass  closely  resembling  white  of  egg.  Sometimes  this  is  so  thick  that  the 
test-tube  containing  it  can  be  inverted  without  spilling  it.  In  alkaline  urine  this 
glairy  mass  is  sometimes  spontaneously  formed. 

The  appearance  of  the  mucus  or  pus  corpuscles  in  urine  depends  largely  on  the 
concentration  of  the  latter.  In  urine  of  low  specific  gravity  the  corpuscles  absorb 
water  and  swell  to  larger  size  than  normal,  while  in  a  highly  concentrated  urine 
they  may  give  out  water  and  become  reduced  in  size  and  shrunken  in  appearance. 

To  recognize  them  under  the  microscope  transfer  a  few  drops  of  the  sediment 
to  a  slide,  and  cover  as  usual.  If  the  nuclei  are  not  distinct  place  a  drop  of 
diluted  acetic  acid  on  the  slide  at  the  edge  of  the  cover  glass.  Part  of  the  acid 
will  flow  under  the  cover  and  mix  with  the  urine.  As  it  does  this  the  clearing 
up  of  the  corpuscles,  with  appearance  of  the  nuclei,  can  be  very  easily  followed. 
Urine  containing  much  pus  is  white  and  milky.  The  same  appearance  is  often 
noticed  with  an  excess  of  earthy  phosphates,  but  the  latter  clear  up  with  acids 
while  the  pus  does  not. 

Epithelium  Cells.  Epithelium  cells  from  different  sources  may  appear  normally 
in  the  urine,  and  the  light  cloud  which  separates  from  normal  urine  on  standing 
consists  chiefly  of  these  cells.  When  present  in  small  amount  this  epithelium 
has  usually  no  clinical  importance,  as  it  easily  finds  its  way  into  the  urine  from 
the  bladder,  vagina,  or  urethra.  An  abundance  of  cells  from  these  organs  would, 
however,  be  considered  pathological,  pointing  to  a  catarrhal  condition. 

Unfortunately,  it  is  not  possible  in  all  cases  to  determine  the  source  of  the  cells, 
as  found  in  urine,  partly  because  cells  from  different  localities  have  frequently 
the  same  general  appearance,  and  partly  because,  owing  to  immersion  in  the  urine, 
they  become  greatly  changed  from  what  they  are  in  the  tissue  as  shown  by  the 
microscopic  study  of  sections.  It  is  customary  to  make  three  rough  divisions  of 
the  cells  as  found  in  the  urine: 

i.  Spherical  cells.     2.  Columnar  or  conical  cells.     3.  Flat  or  scaly  cells. 

The  spherical  cells  are  probably  normally  much  flattened,  but  by  absorption  of 
water  they  become  swollen  and  globular.  These  cells  may  be  derived  from  several 
sources,  as  from  the  uriniferous  tubules  or  from  the  deeper  layers  of  the  lining 
membrane  of  the  pelvis  of  the  kidney,  or  the  bladder,  or  the  male  urethra. 

These  cells  have  a  well-defined  nucleus  resembling  that  of  a  pus  cell.  But  they 
are  much  larger,  and  besides  show  the  nucleus  without  addition  of  acid.  In 
nephritis,  or  other  structural  diseases  of  the  kidney,  these  round  cells  are  found 
along  with  albumin,  and  their  recognition  is  then  a  matter  of  importance  as 
indicating  a  breaking  down  of  the  tubular  walls.  Sometimes  these  cells  form  a 
variety  of  tube  cast,  to  be  described  later.  But  it  must  be  remembered  that  we 
cannot  distinguish  with  certainty  between  the  cells  from  the  tubules  and  those  from 
the  other  localities  mentioned. 

Conical   cells  come  generally   from   the   pelvis   of   the  kidney,    from   the   ureters 


344  PHYSIOLOGICAL    CHEMISTRY. 

and  urethra.  Some  of  these  cells  are  furnished  with  one  or  two  processes,  and  are 
broad  in  the  middle  and  taper  toward  each  end,  while  the  others  are  broad  at  the 
base  and  taper  to  a  point. 

The  large  flat  cells  come  from  the  vagina  or  bladder,  and  it  is  generally  im- 
possible to  distinguish  between  them.  Sometimes  they  are  very  nearly  circular, 
sometimes  irregularly  polygonal  in  outline.  Sometimes  the  vaginal  epithelium  is 
found  in  layers  of  scales,  which  appear  thicker  and  tougher  than  the  cells  from  the 
bladder,  which  occur  singly. 

'  What  was  said  about  the  decomposition  of  blood  or  pus  cells  in  urine  obtains 
also  for  the  various  epithelium  cells.  In  acid  urine  they  may  maintain  their  dis- 
tinct outlines  many  days,  but  in  alkaline  secretion  they  soon  undergo  disintegra- 


W^       ..,,».    ^7^h  \  *-  "^    A-vw:-:?   ^.m. 


Ik      ill  fli  \ 


'■L:~>    ' 


•■•y^y 


t 


90  w%f^m 

Fig.  32.      Common  forms  of  epithelium  scales. 

tion,  which  makes  their  recognition  practically  impossible.  In  general  the  greatest 
importance  attaches  to  the  cells  from  the  tubules  of  the  kidney.  The  presence  of 
albumin  in  more  than  minute  traces  in  the  urine  would  suggest  that  any  smaller 
spherical  cells  present  may  have  had  their  origin  in  the  kidney  rather  than  in  the 
bladder  or  male  urethra.  In  general  it  may  be  said  that  urine  containing  large 
numbers  of  the  smaller,  round  tubule  cells  with  albumin  will  also  show  casts. 

Mucin  Bands.  Urine  containing  much  mucus  sometimes  exhibits  a  deposit 
consisting  of  long  threads  or  bands,  curved  and  bent  in  every  direction.  These 
bands  are  important  because  they  are  sometimes  confounded  with  the  tube  casts 
to  be  described  next.  They  can  be  produced  in  urine  highly  charged  with  mucus 
by  the  addition  of  acids,  and  appear  therefore  sometimes  spontaneously  when  the 
urine  becomes  acid.  These  threads  are  sometimes  covered  with  a  fine  deposit  of 
granular  urates  and  then  bear  some  resemblance  to  granular  casts.  In  general, 
however,  they  are  relatively  longer  and  narrower  than  the  true  casts  of  the 
uriniferous  tubules.  The  mucin  threads  can  occur,  and  frequently  do  occur,  in 
urine  entirely  free  from  albumin,  while  true  tube  casts  are  usually  associated  with 


SOME    PRACTICAL    URINE   TESTS.  345 

albumin,  although  not  always,  as  will  be  explained  below.  The  length  and  shape 
of  the  mucin  threads  may  generally  be  relied  upon  to  distinguish  them  from 
true  casts. 

Casts.  The  structures  properly  termed  casts  are  seldom  found  in  urine  which 
does  not  contain  albumin.  They  are  formed  in  the  uriniferous  tubules,  and,  to  a 
certain  extent,  are  "  casts  "  of  portions  of  the  same.  Their  specific  gravity  differs 
but  little  from  that  of  the  urine,  for  which  reason  they  remain  long  in  suspension. 
It  is  therefore  necessary  to  allow  the  urine  to  stand  some  hours  at  rest,  over 
night  or  longer,  before  attempting  an  examination,  if  a  centrifuge  is  not  at  hand. 

True  casts  of  the  uriniferous  tubules  rarely  appear  in  normal  urine  and  their 
recognition  is  therefore  a  matter  of  the  highest  importance  in  diagnosis.  Much 
has  been  written  on  the  subject  of  the  origin  of  these  bodies  in  the  kidney  and 
several  theories  have  been  advanced  to  account  for  their  formation  and  chemical 
constitution.  Most  of  this  discussion  would  be  out  of  place  in  a  work  like  the 
present  dealing  mainly  with  questions  of  analysis,  but  enough  will  be  given  to  aid 
the  student  in  his  practical  work.  It  must  be  said  that  few  subjects  are  more  per- 
plexing to  the  beginner  than  that  of  their  certain  recognition,  because  of  the  fact 
that  some  varieties  are  so  transparent  as  to  be  almost  invisible,  while  others  are 
closely  resembled  by  formations  of  entirely  different  nature,  not  pathological. 
With  practice,  however,  these  difficulties  can  be  surmounted. 

Most  of  the  bodies  termed  casts  are  formed  of  organized  structures  or  the 
remains  of  such,  but  another  and  rather  common  form  consists  of  crystalline  mat- 
ter, usually  uric  acid  or  fine  granular  urates. 

These  bunches  of  urates  have  no  pathological  significance  and  are  of  frequent 
occurrence.  Urine  containing  them  clears  up  by  heat,  and  the  deposits  themselves 
are  dissipated  by  weak  alkali.  While  it  is  true  that  they  resemble,  to  some  degree, 
the  so-called  granular  casts  referred  to  below,  there  are  certain  well-defined  points 
of  difference.  The  bunches  of  urates  lack  the  coherence  which  can  be  observed 
in  the  true  casts,  and  besides,  the  granulation  is  finer  and  more  clearly  defined. 

The  fact  that  mucin  bands  occasionally  appear  covered  with  a  precipitate  of 
granular  urates  has  been  referred  to.  These  aggregations  are  more  compact  than 
the  loose  bunches  of  urates  just  mentioned  and  much  longer  generally.  They  are 
also  darker  and  therefore  more  easily  seen  than  are  the  casts  proper  or  the  urates. 

The  true  casts  are  made  up  of  matter  in  which  evidence  of  cell  structure  or 
transformation  is  visible.  An  accurate  classification  of  these  bodies  cannot  yet  be 
made,  and,  as  said,  authors  differ  regarding  the  importance  of  several  forms  and 
their  origin.  But  for  our  purpose  it  will  be  sufficient  to  make  the  following  rough 
division,  which  accords  in  the  main  with  what  is  found  in  the  text-books  of  urine 
analysis : 

i.     Blood   casts.  4.     Fatty  casts. 

2.  Epithelium   casts.  5.     Waxy  casts. 

3.  Granular  casts.  6.     Hyaline  casts. 

What  are  termed  blood  casts  consist  of  or  contain  coagulated  blood,  recognized 
by  the  corpuscles.  Plugs  of  this  coagulated  matter  are  forced  out  from  the  tubules 
by  pressure  from  behind,  and  form  one  of  the  most  characteristic  varieties  of  casts. 
They  are  generally  very  dark  in  color,  and  easily  distinguished  from  other  matter. 
A  representation  of  blood  casts  is  given  in  the  following  cut. 

In  epithelium  casts  the  characteristic  substance  is  the  lining  epithelium  of  the 
tubule.  Sometimes  this  lining  epithelium  becomes  detached  in  the  form  of  a  hol- 
low cylinder,  the  walls  consisting  of  the  united  cells.  Again,  the  coagulated  con- 
tents of  the  tubule  in  passing  out  may  carry  the  epithelium  with  it  as  a  coating. 
In  either  case  a  grave  disorder  of  the  kidneys  is  indicated,  as  acute  nephritis,  or 


346  PHYSIOLOGICAL    CHEMISTRY. 

other  disease  in  which  a  profound  alteration  of  the  internal  structure  of  the  organ 
is   involved. 

What  are  termed  granular  casts,  proper,  appear  in  a  variety  of  forms,  produced 
probably  by  the  disintegration  of  blood  or  epithelium  casts. 

There  is  no  uniformity  in  the  fineness  of  the  granulation;  sometimes  a  high 
amplification  is  necessary  to  disclose  the  structure.  Occasionally  blood  corpuscles, 
epithelium,  fat  globules  and  crystals  can  be  detected  in  them,  and  when  derived 
from  blood  cast  disintegration  they  usually  have  a  yellowish  red  color,  which 
makes  their  recognition  comparatively  easy.  In  outline  they  are  generally  regular, 
with  rounded  ends,  one  of  which  is  somewhat  pointed.  Frequently,  however,  they 
appear  to  be  broken,  the  ends  showing  irregular  fracture. 

Fatty  casts  contain  oil   drops  produced  by  some  variety  of   fatty  degeneration 


Fig.  33.     Blood  casts  and  granular  casts. 

of  the  tissues  of  the  kidney.  These  oil  drops  may  form  coherent  bunches,  or  they 
may  be  held  by  patches  of  epithelium.  It  also  happens  that  epithelium  or  granular 
casts  may  be  partially  covered  by  oil  drops.  The  name,  fatty  cast,  is  applied  to 
those  in  which  the  fat  globules  predominate.  Along  with  these  globules  the 
microscope  sometimes  shows  crystals  of  free  fatty  acids,  and  probably  also  of 
soaps  containing  calcium  and  magnesium. 

Waxy  casts  consist  of  the  peculiar  matter  produced  by  amyloid  degeneration 
of  the  kidney.  They  have  a  glistening  wax-like  or  vitreous  appearance,  and  re- 
fract light  very  strongly.  Sometimes  they  reach  a  great  length,  and  they  fre- 
quently are  found  with  blood  corpuscles  or  oil  drops  on  the  surface.  They  have 
been  detected  in  several  renal  disorders.     Illustrations  are  given. 

True  hyaline  casts  are  nearly  transparent  and  hard  to  see  unless  the  illumina- 
tion is  very  carefully  managed.  To  detect  them  it  is  often  necessary  to  add  a 
few  drops  of  a  dilute  solution  of  iodine  in  potassium  iodide  to  the  sediment.  This 
imparts  a  slight  color  which  renders  them  visible. 

The  hyaline  casts  seem  to  be  formed  by  the  passage  of  homogeneous  matter 
from  the  tubules,  leaving  the  epithelium  behind.  A  cast  is  rarely  perfectly  hya- 
line, as  at  least  an  occasional  blood  corpuscle,  fat  globule,  or  epithelium  cell  will 
usually  be  found  attached  to  it.  Waxy  casts  may  be  looked  upon  as  a  special 
form  of  hyaline  casts.     Very  imperfect  representations  are  given  in  the  above  cut. 

In  general,  it  must  be  said  that  the  representation  of  these  casts  on  paper  is 
a  very  difficult  matter.  Ordinarily  they  are  drawn  and  printed  much  too  heavy 
and  dark. 

Hyaline  casts  do  not  necessarily  indicate  kidney  disease,  although  this  is  usually 


SOME    PRACTICAL    URINE   TESTS.  347 

the  case.  They  have  been  found  in  urine  free  from  albumin  and  under  circum- 
stances not  connected  with  renal  disorders. 

The  preservation  of  sediments  containing  casts  is  unusually  difficult  because 
of  the  nature  of  the  material  to  be  preserved.  In  urine  of  the  slightest  alkalinity 
their  disintegration  soon  begins,  so  that  the  outlines  are  rendered  indistinct,  often 
making   identification  impossible. 

For  temporary  preservation  the  addition  of  chloroform  renders  as  good  service 
as  anything  else.  Many  other  sediments  can  be  permanently  mounted  and  kept 
for  future  comparison  but  with  casts  this  can  rarely  be  done. 

Beginners  are  apt  to  overlook  casts  in  their  first  examinations.  It  must  be 
remembered  that  some  of  them  are  nearly  transparent  and  unless  brought  into 
proper  focus  they  may  not  be  seen  at  all.  At  the  outset  students  usually  employ 
too  bright  a  light  in  looking  for  casts.  While  no  specific  directions  can  be  given 
regarding  the  intensity  of   illumination  best  suited  for  the  purpose,  this  may  be 


Fig.  34.      Waxy  and  hyaline  casts. 

said,  that  the  light  commonly  found  necessary  in  studying  ordinary  histological 
slides  is  far  too  bright  to  use  in  the  search  for  casts. 

Practise  alone,  first  under  the  direction  of  the  instructor,  will  indicate  what  is 
proper  here. 

Spermatozoa.  These  minute  bodies,  as  found  in  the  semen  of  man,  have  a 
mean  length  of  about  0.050  millimeter.  Nearly  one-tenth  of  this  is  in  the  head 
portion.  When  observed  in  recently  discharged  semen  they  have  a  characteristic 
spontaneous  movement  by  which  they  are  propelled  forward  rapidly.  This  motion 
is  soon  lost  if  the  semen  is  diluted  with  water  or  similar  liquid.  Hence,  as  usually 
seen  in  urine,  they  are  entirely  motionless.  They  are  found  abundantly  in  the 
urine  of  men  after  coitus  or  nocturnal  emissions,  and  also  in  spermatorrhea,  when 
their  presence   is   continuous   and   characteristic. 

Fungi.  The  urine  sometimes  contains  certain  fungus  growths,  the  recognition 
of  which  is  important.  These  may  have  entered  the  urine  after  voiding,  or  they 
may  have  come  from  the  bladder. 

Normal  urine  when  passed  is  probably  free  from  fungi  of  all  kinds,  but  in  a 
short  time  certain  organisms  enter  it  from  the  air  or  from  other  sources  and  be- 
come active  in  producing  in  it  characteristic  changes. 

The  three  important  groups  of  fungi,  the  schizomycetes  or  bacteria,  the 
hyphomycetes  or  molds,  and  the  blastomycetes  or  yeasts  are  represented  in  the 
organisms  sometimes  found  in  the  urine.  The  conditions  under  which  they  are 
found  will  be  briefly  explained. 


348  PHYSIOLOGICAL    CHEMISTRY. 

Of  the  bacteria  the  following  have  been  observed: 

Micrococcus  Ureae.  This  is  the  exceedingly  common  form  found  in  urine  un- 
dergoing alkaline  fermentation  by  which  urea  is  converted  into  ammonium  carbon- 
ate. It  is  usually  introduced  from  the  air  and  multiplies  very  rapidly  under  ordi- 
nary conditions.  Nearly  all  old  specimens  of  urine,  unless  containing  some  active 
preservative,  are  found  infected  with  this  small  organism.  The  micrococci  are 
minute  spherical  bodies  belonging  to  the  suborder  spherobacteria  and  are  found 
separate  or  in  chains.  They  are  the  smallest  of  the  organized  forms  occurring  in 
urine  and  appear  under  a  power  of  250  diameters  but  little  more  than  points. 

While  generally  finding  their  way  into  urine  after  it  has  been  voided  they  are 


b 


\ 


Fig.  35.      Micrococci  and  other  bacteria. 

occasionally  present  in  the  bladder.  It  is  usually  held  that  under  such  circumstances 
they  have  been  introduced  by  a  dirty  catheter  or  sound,  although  cases  are  on  record 
where  this  has  not  been  proved. 

In  the  bladder  they  give  rise  to  alkaline  fermentation,  so  that  the  voided  urine 
may  show  ammonium  carbonate  directly. 

It  is  now  recognized  that  the  production  of  ammonium  carbonate  from  urea  may 
be  brought  about  by  several  species  of  bacteria. 

Streptococcus  Pyogenes  is  a  pathogenic  form  sometimes  found  in  the  urine  in 
cases  of  infectious  diseases. 

Sarcinae.  The  genus  sarcina  is  frequently  classed  with  the  spherobacteria  and 
several  species  have  been  found  in  urine.  The  cells  are  larger  than  those  of 
micrococcus  urese,  and  are  arranged  in  groups  of  two  or  four  usually.  They  are 
not  pathogenic. 

Bacilli.  Several  species  of  the  genus  bacillus  are  found  in  urine  in  disease. 
The  most  important  of  these  are  the  typhoid  bacillus,  bacillus  typhi  abdominalis, 
the  tubercle  bacillus,  bacillus  tuberculosis,  and  the  bacillus  of  glanders,  bacillus 
mallei.  These  bacilli  occur  in  urine  only  during  the  progress  of  the  correspond- 
ing diseases  and  their  detection  is  of  the  highest  interest.  A  description  of  the 
methods  to  be  followed  for  the  certain  demonstration  of  these  bodies  is  not  within 
the  scope  of  this  book,  but  must  be  looked  for  in  the  laboratory  manuals  of 
bacteriology.  It  may  be  said,  in  general,  that  in  diseases  characterized  by  the 
presence  of  certain  bacteria  in  the  blood,  they  may  be  found  also  in  the  urine. 

Spirilla.  Certain  species  of  the  genus  spirillum  have  been  found  in  urine.  The 
best  known  of  these  is  the  spirillum  of  relapsing  fever,  spirillum  obermeieri. 
This  is  only  found  rarely  and  as  its  habitat  is  the  blood  of  relapsing  fever  patients 
it  must  enter  the  urine  through  a  hemorrhage  into  the  kidney.  Its  form  is  that  of 
a  long,  wavy  spiral,  which  makes  its  detection  somewhat  easy. 

Although  not  pathogenic  it  is  well  to  call  attention  to  certain  molds  which  may 
sometimes  be  seen  in  urine.     The  common  blue-green  mold,  penicillium  glaucum, 


SOME    PRACTICAL    URINE    TESTS. 


349 


is  the  best  known  of  these,  and  is  occasionally  found  in  urine  along  with  yeast 
cells.  Another  mold  which  has  been  found  in  urine  is  the  oidium  lactis,  commonly 
occurring  in  milk  and  butter.  It  has  been  observed  in  fermenting  diabetic  urine. 
Both  of  these  fungi  enter  the  urine  after  voiding.  In  urine  which  has  stood  some 
time  in  a  cool  place  the  penicillium  glaucum  sometimes  becomes  covered  with 
an  incrustation  of  urates  or  minute  crystals  of  uric  acid. 

Finally   we   have   yeast   cells   in   urine   and   sometimes   in   great   numbers.     Like 
other  fungi  they  enter  the  urine   from  the  air  and  when  not  very  abundant  have 


Fig.  36.      Yeast  cells  and  common  mold. 

no  significance.  In  great  numbers  the  yeast  cells  suggest  presence  of  sugar.  The 
ordinary  yeast  plant,  saccharomyccs  cerevisice,  is  shown,  isolated  and  budding,  in 
the  accompanying  figure.  These  forms  are  described  here  because  their  presence 
is  often  confusing  to  the  beginner. 


UNORGANIZED    SEDIMENTS. 

Uric  Acid.  Among  the  more  common  of  the  unorganized  sediments  found  in 
urine  this  must  be  mentioned  first.  As  was  explained  in  the  last  section  uric  acid 
occurs  normally  in  combination  in  all  human  urine. 

Some  time  after  its  passage  urine  often  undergoes  what  has  been  spoken  of 
as  the  acid  fermentation  by  which  a  precipitate  of  urates  and  even  free  uric  acid 


Fig.  37.      Uric  acid. 


may  appear.  This  reaction  is  in  no  case  due  to  a  ferment  process  in  the  ordinary 
sense  of  the  term,  but  is  probably  brought  about  by  a  purely  chemical  double 
decomposition.  Urine  contains  acid  sodium  phosphate  and  neutral  sodium  urate 
and  it  has  been  suggested  that  these  react  on  each  other  according  to  the  follow- 
ing equation : 

Na2CBH3N.O,  +  NaH2PO<  =  NaC.H.N.0,  +  Na2HPO«. 


3  SO  PHYSIOLOGICAL    CHEMISTRY. 

The  precipitate  of  acid  urate  settles  out  and  forms  a  light  reddish  deposit.  If 
the  amount  of  acid  phosphates  present  is  excessive  the  reaction  may  go  still  further, 
resulting  in  the  precipitation  of  free  uric  acid.  The  well-characterized  crystals 
of  uric  acid  are  often  found  with  the  sediment  of  fine  urates.  Sometimes  this 
liberation  and  precipitation  of  the  acid  takes  place  in  the  bladder,  and  the  urine,  as 
passed,  shows  the  crystals  or  "  gravel."  If  they  are  relatively  large,  which  is 
sometimes  the  case,  their  passage  through  the  urethra  may  cause  severe  pain. 

As  the  illustrations  show,  uric  acid  occurs  in  a  great  variety  of  forms.  The 
rosettes  and  whetstone-shaped  crystals  are  probably  the  most  common,  while  long 
spiculated  forms  are  frequently  seen.  Pure  uric  acid  is  colorless  but  as  deposited 
from  urine  it  is  always  reddish  yellow,  because  of  its  property  of  carrying  down 
coloring-matters.  The  crystals  are  often  so  large  that  their  general  form  can  be 
seen  by  the  naked  eye ;  usually,  however,  they  are  minute. 

Uric  acid  crystals  when  once  deposited  are  not  readily  redissolved  by  heat,  but 
they  go  into  solution  by  the  addition  of  alkali.  If  the  urine  contains  extraneous 
matter,  as  specks  of  dust,  bits  of  hair,  cotton  or  wool  fibers,  the  crystals  are  very 
apt  to  deposit  on  them. 

Urates.  The  common  fine  sediments  of  urine  are  usually  urates  or  amorphous 
phosphates.  They  can  be  most  readily  distinguished  by  their  behavior  with  acids 
and  on  application  of  heat.     Urates   disappear  on  warming  the   urine  containing 


Fig.  38.      Common  crystalline  and  granular  urates. 

them,  while  a  phosphate  sediment  is  rendered  more  abundant.  A  urate  sediment 
is  little  changed  by  acids,  while  the  phosphates  dissolve  completely  if  the  urine 
is  made  acid  in  reaction  with  hydrochloric  or  nitric  acid.  The  acid  urates  of 
sodium  and  ammonium  are  the  most  abundant  and  are  shown  in  the  cut.  Acid 
ammonium  urate  may  exist  in  urine  which  has  become  alkaline  from  the  decomposi- 
tion of  urea  and  formation  of  ammonium  carbonate,  and  may  therefore  be  seen  in 
company  with  the  phosphate  sediments.  The  other  urates  dissolve  in  alkaline  urine. 
Like  uric  acid  the  urates  appear  in  a  great  variety  of  forms,  and  there  is  still 
some  uncertainty  about  the  composition  of  some  of  their  crystals  which  have  been 
found  in  urine. 

Leucine  and  Tyrosine.  These  two  substances  are  of  rare  occurrence  in  urine 
and  appear  only  under  pathological  conditions.  Urine  containing  them  shows 
usually  strong  indications  of  the  presence  of  biliary  matters  as  they  generally  are 
found  in  consequence  of  some  grave  disorder  of  the  liver  in  which  destruction 
of  its  tissue  is  involved.  They  have  been  most  frequently  found,  and  associated, 
in  acute  yellow  atrophy  of  the  liver  and  in  severe  cases  of  phosphorus  poisoning. 
In   general   they  must  be  considered  as   products   of   disintegration   and   are   pro- 


SOME    PRACTICAL   URINE    TESTS. 


351 


duced  in  the  intestine  in  large  quantity  by  bacterial  agency  in  the  last  stages  of 
the  digestion  of  proteins,  as  was  pointed  out  in  an  early  chapter. 

As  both  bodies  are  slightly  soluble  they  may  not  be  seen  directly,  but  only  after 
partial  concentration  of  the  urine.  In  pure  condition  leucine  crystallizes  in  thin 
plates  but  from  urine  it  separates  in  spherical  bunches  made  up  of  fine  plates  or 
needles.  These  bunches  are  sometimes  so  compact  that  it  is  hard  to  distinguish 
between  them  and  other  substances,  particularly  lime  soaps  and  oil  drops.  Chem- 
ical tests  must  therefore  be  applied.  If  mercurous  nitrate  is  added  to  a  leucine 
solution  and  the  mixture  is  warmed,  metallic  mercury  precipitates.  This  test  can 
be  carried  out  only  when  the  substance  is  abundant  enough  to  be  purified  by  crys- 
tallization from  hot  water.  Pure  leucine,  when  strongly  heated  with  nitric  acid 
on  platinum,  forms  a  colorless  residue,  which  when  heated  with  potassium  hydrox- 
ide leaves  an  oil-like  drop  that  does  not  wet  the  platinum. 

Tyrosine  is  usually  seen  in  long  needles,  which  sometimes  are  bunched  in  the 
form  of  sheaves,  and  is  more  readily  recognized  than  is  leucine.  Tyrosine  heated 
with  nitric  acid  on  platinum  turns  orange-yellow,  and  leaves  a  dark  residue  which 
becomes  reddish  yellow  by  addition  of  caustic  alkali.  Solutions  containing  tyrosine 
when  treated  hot  with  mercuric  nitrate  and  potassium  nitrite,  turn  red  and  finally 
throw  down  a  red  precipitate. 

Cystin.  This  is  a  rare  sediment,  although  it  is  found  constantly  in  the  urine 
of  certain  individuals.  It  crystallizes  in  thin  hexagonal  plates,  small  ones  some- 
times resting  upon  or  overlapping  large  ones.    The  crystals  are  regular  in  form  but 


Fig.  39.     Leucine  spheres,  tyrosine  needles  and  cystin  plates. 


variable  in  size  and  readily  recognized.  A  rare  form  of  uric  acid  crystallizes  in  a 
somewhat  similar  manner  but  the  two  substances  differ  in  their  behavior  toward 
ammonia.  To  distinguish  between  them  in  the  microscopic  test  place  a  drop  of 
ammonia  water  on  the  slide  and  allow  it  to  pass  under  the  cover  glass.  Cystin 
dissolves  but,  unless  heated,  uric  acid  does  not.  When  the  ammonia  evaporates 
cystin  precipitates. 

Cystin  is  precipitated  from  urine  by  addition  of  acetic  acid.  Mucin  and  uric 
acid  may  come  down  at  the  same  time.  The  precipitate  is  collected  on  a  filter, 
washed  with  water  and  finally  dissolved  in  ammonia.  By  neutralizing  the  am- 
moniacal  filtrate  with  acetic  acid  and  concentrating  a  little,  it  comes  down  in  the 
characteristic  form  suitable  for  microscopic  recognition. 

Fat  Globules.  These  are  often  seen  in  urine,  but  in  most  cases  have  not  been 
voided  with  it.  They  can  come  from  several  extraneous  sources,  as  from  a  cathe- 
ter, from  vessels  in  which  the  urine  is  collected  or  sent  for  examination,  from  ad- 
mixed sputum,  etc.,  which   facts  should  be  borne  in  mind. 


352  PHYSIOLOGICAL    CHEMISTRY. 

Fat  has  been  found  in  cases  of  fatty  degeneration  of  the  kidney  and  more 
abundantly  in  chyluria  where  communication  seems  to  be  formed  between  the 
lymphatics  and  the  urinary  tract  by  the  invasion  of  small  thread  worms. 

Hippuric  Acid.  This  acid  is  found  normally  in  human  urine  in  small  amount, 
It  may  be  found  in  large  quantity  after  taking  benzoic  acid  and  may  even  appear 
in  crystalline  form  in  the  sediment.     It  has  no  pathological  importance,  ordinarily. 

Calcium  Carbonate.  This  is  sometimes  observed  as  a  coarse,  granular  sedi- 
ment which  dissolves  with  effervescence  in  acetic  acid.  It  occasionally  forms 
dumb-bell  crystals,  and  is  devoid  of  pathological  importance. 

Calcium  Sulphate.  Crystals  of  this  substance  are  rarely  found  in  urine.  They 
form  long,  colorless  needles,  or  narrow,  thin  plates. 

Calcium  Oxalate.  We  have  here  one  of  the  commoner  of  the  crystalline  bod- 
ies observed  in  urine. 

This  may  be  found  in  neutral  or  alkaline  urine,  but  more  commonly  in  that 
of  acid  reaction.  It  occurs  normally  and  sometimes  is  very  abundant,  especially 
after  the  consumption  of  vegetables  containing  oxalic  acid. 

Two  principal  forms  of  the  crystals  are  found,  the  octahedral  and  dumb-bell 
crystals. 

The  octahedra  have  one  very  short  axis  which  gives  the  crystals  a  flat  appear- 
ance.    When   seen   with  the   short  axis   perpendicular  to   the  plane   of   the  cover 


Fig.  40.      Calcium  oxalate. 


glass,  which  is  the  common  position,  they  appear  as  squares  crossed  by  two  bright 
lines.  Sometimes  they  are  seen  on  edge,  and  then  present  a  rhomb  in  section 
with  one  diameter  very  much  shorter  than  the  other. 

A  form  of  triple  phosphate  bears  a  slight  resemblance  to  calcium  oxalate,  but 
it  is  soluble  in  acetic  acid,  while  the  oxalate  is  not. 

The  dumb-bells  are  much  less  common  than  the  octahedra,  and  are  found  in 
several  modified  forms,  as  shown  in  one  of  the  figures. 

The  clinical  significance  of  the  oxalate  is  not  clearly  understood.  It  does  not 
seem  to  be  characteristic  of  any  disease  even  when  occurring  in  quantity.  It  has 
been  found  considerably  increased  in  dyspeptic  conditions,  but  not  always,  and 
many  of  the  statements  found  concerning  its  significance  seem  to  have  been  based 
on  insufficient  observations. 

Urine  may  contain  a  large  amount  of  oxalic  acid,  which  does  not  show  as  a 
sediment,  but  must  be  found  by  precipitation  with  calcium  chloride  in  presence  of 
ammonium  hydroxide.  Acetic  acid  is  then  added  in  very  slight  excess  and  the 
mixture  is  allowed  to  stand  for  precipitation. 


SOME    PRACTICAL    URINE    TESTS. 


353 


The  constant  or  prolonged  excretion  of  large  amounts  of  oxalic  acid  is  spoken 
of  as  oxaluria. 

The  Phosphates.  It  has  been  explained  that  phosphates  of  alkali  and  alkali- 
earth  metals  occur  normally  in  the  urine,  and  a  method  was  given  for  their  esti- 
mation. As  sediment  we  know  several  forms  of  calcium  and  magnesium  phos- 
phates and  the  microscopic  detection  of  these  will  be  here  explained.  In  normal 
fresh  urine  of  acid  reaction  these  phosphates  are  held  in  solution,  but  if  the  urine 
as  passed  is  alkaline  it  is  often  turbid  from  the  presence  of  basic  phosphates  held 
in  suspension.  Urine  which  has  stood  long  enough  to  undergo  the  alkaline  fer- 
mentation always  contains  phosphates  in  the  sediment.  Finally,  it  must  be  re- 
membered that  a  neutral  or  very  slightly  acid  urine,  containing  ammonium  salts 
in  abundance,  may  also  deposit  a  crystalline  precipitate  of  ammonium  magnesium 
phosphate.     The  common  phosphate  sediments  are  those  consisting  of  ammonium 


Fig.  41.      Triple  phosphate. 


magnesium  phosphate  (triple  phosphate),  normal  magnesium  phosphate,  neutral 
calcium  phosphate,  and  mixed  amorphous  phosphates  of  calcium  and  magnesium. 

Triple  Phosphate.  Of  the  crystalline  phosphate  deposits  this  is  the  most  abun- 
dant and  at  the  same  time  the  most  characteristic. 

The  crystals  are  the  largest  found  in  urine,  and  from  their  shape  are  some- 
times spoken  of  as  coffin-lid  crystals.  Ordinarily  they  are  not  found  in  perfectly 
fresh  urine,  but  after  it  has  undergone  the  alkaline  fermentation  they  are  gen- 
erally present  in  profusion. 

Normal  Magnesium  Phosphate.  Crystals  having  the  composition,  Mg3(POJ2. 
22FLO,  are  sometimes  found  in  urine  of  nearly  neutral  reaction.  They  consist 
of  thin,  transparent,  rhombic  plates  with  angles  approximately  6o°  and  1200.  If 
urine  containing  this  sediment  becomes  alkaline,  triple  phosphate   forms. 

Neutral  Calcium  Phosphate.  This  has  the  composition,  CaHPOv2H20,  and 
is  found  in  urine  of  neutral  or  slightly  acid  reaction.  It  crystallizes  frequently 
in  rosettes  formed  of  wedge-shaped,  single  crystals,  uniting  at  their  apices.  The 
cut  shows  some  variations  in  the  form. 

Amorphous  Phosphates.  Finally  we  have  the  very  common,  finely  granular, 
earthy  phosphates  in  amorphous  condition.  This  sediment  dissolves  readily  in 
weak  acetic  acid  and  is  colorless.  The  common  amorphous  urate  sediment  is  col- 
ored and  does  not  dissolve  in  acetic  acid.  On  addition  of  sodium  carbonate  or 
hydroxide  to  urine,  the  precipitate  which  forms  consists  mainly  of  this  phosphate. 

These  several  phosphates  can  be  produced  artificially  and  should  be  made  for 
study   and   comparison.     The  normal   magnesium   phosphate  can  be  made  by   dis- 

24 


354 


PHYSIOLOGICAL    CHEMISTRY. 


solving  15  grams  of  crystallized  common  sodium  phosphate  in  200  cc.  of  water 
and  mixing  this  with  3.7  grams  of  crystallized  magnesium  sulphate  in  2000  cc. 
of  water.  Enough  sodium  bicarbonate  is  added  to  give  an  amphoteric  reaction 
and  then  the  mixture  is  allowed  to  stand  a  day  or  more  for  precipitation. 

Crystals  of  triple  phosphate  of  peculiar  form  are  often  obtained  by  adding 
ammonia  to  urine,  and  sometimes  a  trace  of  ammonia  is  sufficient  to  throw  down 
the   crystals   of   neutral   calcium   phosphate.     The   latter   can  also   be   obtained  by 


Fig.  42.     Neutral  calcium  phosphate  and  amorphous  phosphate. 

adding  to  a  weak  solution  of  crystallized  sodium  phosphate  a  trace  of  acid  and 
then  a  very  little  calcium  chloride  solution. 

URINARY    CALCULI. 

Calculi,  like  the  sediments  just  described,  are  formed  by  the  precipitation  of 
certain  substances  from  the  urine,  but  in  compact  form.  Occasionally  a  calcu- 
lus consists  of  a  single  substance,  as  calcium  oxalate  or  cystin,  but  in  the  great 
majority  of  cases  a  mixture  of  bodies  is  present,  these  being  deposited  usually 
in  layers  around  a  nucleus  which  serves  as  the  foundation  of  the  concretion. 
Calculi  are  built  up  much  as  certain  forms  of  crystals  are  by  successive  depositions 
on  a  nucleus.  Uric  acid  is  a  very  common  nucleus  on  which  may  be  deposited 
urates,  phosphates,  organic  matters,  etc. 

Calculi  are  sometimes  distinguished  as  primary  or  secondary.  Primary  cal- 
culi may  be  traced  to  an  alteration  of  the  urine  of  such  a  nature  that  its  reaction 
is  constantly  acid.  The  foundation  for  the  concretions  in  this  case  is  found  in 
the  kidney  and  they  are  built  up  of  such  substances  as  most  easily  deposit  from 
acid  urine.  Secondary  calculi  are  generally  formed  in  the  bladder,  and  have  for 
nuclei  matters  precipitated  from  alkaline  urine,  as  coagulated  blood  or  other 
organic  substances.  Sometimes  fragments  introduced  into  the  bladder  from  with- 
out serve  as  the  foundation  for  these  secondary  formations.  Bits  of  catheters, 
remains  of  bougies,  and  other  things  have  been  found  as  the  nuclei  around  which 
concretions  have  formed.  The  recognition  of  the  nucleus  is  a  matter  of  the  first 
importance  as  this  gives  a  clew  to  the  determining  cause  active  in  the  formation 
of  the  calculus. 

In  making  an  examination,  then,  of  a  calculus,  it  is  first  cut  in  two  by  means 
of  a  very  sharp  thin  saw.  This  exposes  the  nucleus  which  may  often  be  recog- 
nized by  the  eye  alone.  If  one  of  the  halves  be  polished  it  is  often  possible  to 
discern  distinctly  the  various  layers  grouped  around  the  center. 

In  a  large  number  of  cases  examined  by  Ultzmann  about  80  per  cent,  were 
found  to  contain  uric  acid  as  the  nucleus. 

Chemical    Examination.    In   the   chemical    examination   of   a   calculus    several 


SOME    PRACTICAL    URINE    TESTS.  355 

methods  may  be  employed.  We  may  begin  by  applying  certain  preliminary  tests 
designed  to  show  the  general  nature  of  the  stone. 

Heat  Test.  Reduce  some  of  the  calculus  to  a  powder  and  heat  to  bright  red- 
ness on  platinum  foil.  Two  cases  may  arise:  (a)  the  powder  is  completely  con- 
sumed; (b)  the  powder  is  only  partially  consumed  or  not  at  all. 

Case  (a).  If  this  is  the  result  of  the  incineration  the  following  substances 
may  be  suspected : 

Uric  Acid,  which  may  be  recognized  by  dissolving  a  little  of  the  powder  in 
weak  alkali,  precipitating  by  hydrochloric  acid  and  examining  the  precipitate  by 
the  microscope. 

Ammonium  Urate.  This  gives  the  above  reaction  under  the  microscope,  and 
is  further  recognized  by  the  liberation  of  ammonia  when  heated  with  a  little  pure 
sodium  hydroxide  solution. 

Cystin.  Dissolve  some  of  the  powder  in  ammonia,  filter  if  necessary  and  allow 
drops  of  the  filtrate  to  evaporate  spontaneously  on  a  slide.  Cystin  is  then  recog- 
nized by  the  microscope  as  already  explained.  Cystin  contains  sulphur  which, 
on  burning  on  the  platinum  foil,  gives  rise  to  a  disagreeable  sharp  odor.  If  a 
little  of  the  powder  be  heated  with  a  mixture  of  potassium  nitrate  and  sodium 
carbonate  the  sulphur  is  oxidized  to  sulphate,  which  may  be  recognized  by  the 
usual  tests. 

Xanthine.  This  is  a  rare  substance  in  calculi.  Those  consisting  wholly  of 
xanthine  are  brown  in  color  and  take  a  wax-like  polish. 

Organized  Matter.  Parts  of  blood  cells,  epithelium,  precipitated  mucin,  pus 
corpuscles  and  similar  substances  may  become  entangled  with  the  growing  stone 
and  even  form  a  large  part  of  it.  On  burning,  these  bodies  are  recognized  by 
the  characteristic  odor  of   nitrogenous  matter. 

Case  (b).  When  an  incombustible  residue  is  left  on  the  platinum  foil  the  stone 
may  contain  the  following  constituents  : 

Calcium  Oxalate.  Stones  of  this  substance  are  very  hard  and  break  with  a 
crystalline  fracture.  They  are  often  called  "  mulberry  calculi."  When  the  powder 
is  heated  it  decomposes,  leaving  carbonate,  which  may  be  recognized  by  its  effer- 
vescence with  acids. 

Calcium  and  Magnesium  Phosphates.  They  leave  a  residue  in  which  the  metals 
and  phosphoric  acid  may  be  detected  by  simple  tests  of  qualitative  analysis.  The 
ignited  powder  is  soluble  in  hydrochloric  acid  without  effervescence.  When  am- 
monia is  added  to  this  solution  in  quantity  sufficient  to  give  an  alkaline  reaction, 
a  precipitate  of  triple  phosphate  or  calcium  phosphate  appears,  which  may  be  recog- 
nized by  the  microscope. 

The  above  tests  are  generally  sufficient  to  tell  all  that  is  practically  necessary 
about  the  calculus.  If  more  detailed  information  is  desired  a  systematic  analysis 
must  be  made. 


CHAPTER   XXI. 

THE  GASEOUS  EXCRETION.    RESPIRATION. 

In  the  last  chapters  the  amount  of  nitrogen  excreted  with  the  urine 
was  discussed  at  some  length.  With  the  nitrogen  certain  correspond- 
ing proportions  of  carbon,  hydrogen  and  oxygen  are  excreted  in  the 
urea,  uric  acid  and  other  bodies  described.  But  the  larger  amounts  of 
these  elements  are  thrown  off  from  the  body  in  different  form,  and 
especially  in  the  carbon  dioxide  and  water  vapor  eliminated  in  respira- 
tion and  perspiration.  From  certain  classes  of  foods  the  end  products 
formed  are  these  two  only  when  the  oxidation  is  ideally  complete. 
This  is  the  case  with  the  fats  and  carbohydrates,  and  supposing  them 
wholly  burned  in  the  body  the  final  results  are  represented  in  this  way, 
taking  typical  substances  for  illustration : 

CH^O,  +  602  =  6C02  +  6H  20, 

C3H5(C18H3502)3  +  163O  =  57C02  +  S5H20. 

In  the  actual  behavior  of  these  compounds  in  the  human  body,  how- 
ever, the  results  are  somewhat  different.  The  oxidation  is  never  quite 
as  complete  as  here  indicated,  as  traces  of  both  carbohydrates  and  fats 
are  left  in  more  complex  forms. 

THE   RESPIRATORY    QUOTIENT. 

In  studying  the  completeness  of  oxidation  of  certain  foods  much 
has  been  learned  by  a  consideration  of  the  factor  known  as  the  respira- 
tory quotient  which  is  simply  the  ratio  of  the  carbon  dioxide  elimi- 
nated to  the  oxygen  absorbed,  measured  by  volume.  This  quotient  is 
therefore  given  by  the  expression  C02/02.  For  the  sugar  of  the 
above  equation  we  require  six  molecules  of  oxygen,  and  the  carbon 
dioxide  produced  is  also  six  molecules.  Hence  C02/02  =  1.  For  all 
common  carbohydrates  the  result  is  the  same.  For  the  fats,  however, 
the  quotient  is  much  smaller  since  57  C02  is  the  carbon  dioxide  volume 
excreted  for  an  oxygen  consumption  of  81.5  02.  In  this  case  C02/02 
=  57/81.5  =  0.7.  For  the  protein  bodies  the  factor  cannot  be  as 
easily  calculated,  since  we  are  not  able  to  assign  a  formula  to  these 
substances,  and  moreover  we  are  not  familiar  with  all  their  oxidation 
products.  But  from  the  percentage  composition,  and  the  known  facts 
regarding  the  elimination  of  urea,  uric  acid,  ammonia  and  creatinine 

356 


THE   GASEOUS    EXCRETION.  357 

it  is  possible  to  calculate  an  approximate  quotient.  This  is  about  0.8, 
which  factor  may  be  used  in  calculations. 

The  use  of  these  quotients  is  ordinarily  based  on  the  assumption 
that  the  oxidation  is  a  direct  one,  and  that  corresponding  to  the  oxygen 
absorbed  there  is  almost  immediately  a  liberation  of  carbon  dioxide  in 
the  right  proportion.  But  this  assumption  does  not  hold  absolutely 
true;  the  breakdown  of  carbohydrate,  for  example,  may  yield  at  first, 
in  part,  products  with  high  oxygen  content,  from  which  C02  separates 
later.  In  other  words,  there  may  be  an  apparent  temporary  storing 
up  of  oxygen,  which  would  make  the  quotient  appear  low.  Later  a 
compensating  excessive  liberation  of  carbon  dioxide  would  have  the 
opposite  result.  However,  in  observations  carried  out  through  a  period 
of  proper  length  these  variations  would  not  affect  the  general  mean. 

The  Carbon  and  Nitrogen  Balance.  The  body  is  in  carbon  equi- 
librium when  just  as  much  carbon  is  eliminated  as  is  consumed  in  the 
food,  and  a  determination  of  this  element  in  the  various  excreted 
products  and  in  the  food  is  sufficient  to  show  whether  there  is  gain, 
loss  or  equilibrium  in  body  weight.  All  the  food  stuffs  are  organic,  it 
will  be  remembered,  and  contain  carbon  as  the  fundamental  element. 

A  change  in  weight  may  result  from  gain  or  loss  in  fat  or  gain  or 
loss  in  protein.  Nitrogen  equilibrium  exists  when  income  and  outgo 
are  equal ;  in  this  case  all  the  proteins  consumed  as  foods  are  decom- 
posed. A  determination  of  nitrogen  in  the  urine  and  feces,  coupled 
with  a  knowledge  of  the  food  protein,  will  decide  this  point,  since  the 
excreted  nitrogen  multiplied  by  6.25  gives  a  measure  of  the  food  pro- 
tein. The  most  accurate  method  of  reaching  the  value  of  the  excreted 
nitrogen  is  by  Kjeldahl  determinations  on  the  urine  and  feces,  but  good 
approximate  results  are  secured  by  determination  of  urea  alone,  it 
being  remembered  that  about  85  per  cent  of  the  urinary  nitrogen 
appears  in  this  form. 

Respiration  Apparatus.  To  determine  the  volume  of  oxygen  in- 
haled and  carbon  dioxide  given  off,  the  animal  or  person  under  experi- 
ment is  placed  in  a  respiration  chamber  of  some  kind.  In  a  form  of 
respiration  chamber  sometimes  used,  an  accurately  measured  volume 
of  air  with  known  content  of  moisture  and  carbon  dioxide  is  forced 
through.  The  air  leaving  the  tight  chamber  is  analyzed  and  the 
amount  of  carbon  dioxide,  moisture  and  oxygen  determined.  This 
last  determination  may  be  made  directly,  or  the  loss  of  oxygen  by  the 
respiration  of  the  person  in  the  cage  may  be  found  by  calculation  from 
this  basis:  The  sum  of  all  the  factors  consumed,  that  is  the  food  and 
the  oxygen,  plus  the  body  weight,  must  be  balanced  by  the  weight  of 


3  5§  PHYSIOLOGICAL    CHEMISTRY. 

the  body  at  the  end  of  the  experiment  plus  the  various  excreted  matters. 
HA  represents  the  body  weight  at  the  beginning  of  the  test  and  A' 
the  body  weight  at  the  end  of  the  test,  Ox  the  oxygen  consumed,  F  the 
food  consumed,  Ex  the  total  excreta  by  weight,  then 

A  +  Ox  +  F  =  A'  +  Ex. 

Ox  =  A'  +  Ex—(A  +  F). 

In  some  of  the  recent  forms  of  respiration  apparatus,  especially  that 
of  Atwater  and  Rosa,  extremely  accurate  results  are  possible  in  the 
determination  of  carbon  dioxide  and  moisture  produced;  but  with 
increase  in  size  of  the  apparatus  a  direct  determination  of  oxygen  dif- 
ference becomes  more  and  more  difficult. 

In  the  Zuntz  apparatus,  which  is  often  used  for  short  experiments 
on  the  gaseous  excretion  only,  a  peculiar  mouthpiece  is  worn  which 
permits  a  collection  of  the  carbon  dioxide  and  vapor  from  the  lungs, 
and  of  the  total  expired  air.  A  determination  of  the  oxygen  and 
carbon  dioxide  is  accurately  made  and  this  furnishes  all  the  data  nec- 
essary for  the  calculation.  The  nose  is  closed  in  this  experiment;  the 
mouthpiece  is  so  arranged  that  air  may  be  drawn  in  without  allowing 
the  excretory  products  to  escape. 

DEDUCTIONS  FROM  RESPIRATION  EXPERIMENTS. 

These  are  undertaken  to  answer  a  number  of  important  questions. 
The  weight  of  carbon  dioxide  excreted  may  reach  fifteen  hundred 
grams  or  more  daily  and  it  is  interesting  to  know  under  what  circum- 
stances it  is  increased  and  when  diminished.  Very  simple  observa- 
tions show  that  the  body  at  rest  produces  much  less  of  the  gas  than 
does  the  body  at  work.  In  the  latter  condition  the  destruction  of  food 
stuffs  is  called  for  to  liberate  mechanical  energy.  This  is  practically 
possible  only  through  oxidation,  and  carbon  dioxide  is  the  first  tangible 
result  of  the  oxidation. 

The  question  also  comes  up,  what  kind  of  organic  matter  is  most 
readily  or  most  commonly  oxidized  when  work  is  done  ?  On  this  ques- 
tion much  has  been  written  and  our  views  have  undergone  various 
changes  through  the  years.  Liebig  considered  the  proteins  as  the 
foods  which  must  be  burned  to  enable  us  to  do  mechanical  work,  but 
in  a  famous  experiment  by  Fick  and  Wislicenus,  undertaken  to  throw 
some  light  on  this  question,  no  great  excess  in  the  excretion  of  urea 
was  found  in  the  work  of  ascending  the  Faulhorn,  and  the  protein 
oxidized  was  far  too  little  to  account  for  the  work  done.  Other  inves- 
tigators reached  the  same  conclusion,  but  it  has  been  found  that  under 


THE    GASEOUS    EXCRETION. 


359 


certain  conditions  the  proteins  may  be  consumed  to  do  work.  Ordi- 
narily fats  and  carbohydrates  are  used  in  preference,  and  no  large 
amount  of  protein  is  used  if  the  other  substances  are  present  in  suffi- 
cient quantity. 

The  question  of  what  kind  of  foodstuff  is  oxidized  through  periods 
of  work  and  rest  may  be  answered  by  experiment.  As  just  intimated, 
examinations  of  the  urine  give  us  information  as  to  the  nitrogen  excre- 
tion, and  the  extent  of  oxidation  of  fats  and  carbohydrates  may  be 
measured  by  respiration  experiments.  In  a  fasting  animal  at  rest  the 
respiratory  quotient  sinks  to  a  value  but  little  above  0.7,  showing  that 
the  substance  metabolized  is  mainly  fat ;  as  some  proteins  are  also  used 
up  the  quotient  cannot  absolutely  reach  0.7.  If  work  is  done  by  the 
fasting  animal,  the  carbohydrate  bodies  of  the  muscular  juices,  glyco- 
gen essentially,  are  called  upon  and  their  effect  is  added  to  that  of  the 
proteins  in  raising  the  respiratory  quotient.  On  the  other  hand,  it  has 
been  found  that  a  well-fed  animal  at  rest,  with  abundance  of  carbo- 
hydrates in  the  ration,  will  excrete  a  volume  of  carbon  dioxide  nearly 
as  great  as  that  of  the  oxygen  absorbed.  In  this  case  the  ratio  C02/02 
shows  that  essentially  carbohydrates  are  burned  and  that  fat  is  allowed 
to  accumulate.  When  very  hard  work  is  done  by  the  well-nourished 
animal  the  quotient  sinks  to  an  intermediate  value,  showing  that  fats 
are  now  consumed  as  well.  This  would  be  evident  also  from  observa- 
tions continued  over  a  long  period  in  which  no  accumulation  of  fat 
could  be  recorded.     With  moderate  work  there  is  not  much  change. 

Some  of  these  results  are  illustrated  by  the  figures  in  the  following 
table  taken  from  observations  published  by  Chauveau  and  Laulanie,  in 
which  dogs  were  the  subjects  of  experiment.  These  figures  show 
very  clearly  alteration  in  the  respiratory  quotient  with  work,  and  also 
by  diet. 


Food  Consumption. 

CO 

Observed  Ratio        2  ,  or  Respiratory  Quotient. 

o2 

No. 

mi* 

Minutes  of  Work   Before 
Observations. 

Minutes  of  Rest  Follow- 
ing Work. 

30 

45 

60 

90 

120 

180 

45 

60 

120 

240 

I 

2 

3 
4 

! 

24  hours  fast. 
6  days  fast. 

1  day  fast. 

2  days  fast. 

3  days  fast. 
After  full  meal. 
After  full  meal. 

0.790 
0.750 
0.874 
0.740 
0.685 
1033 
1. 000 

0.943 
O.819 

I.OI7 

O.905 
O.84O 
O.895 
O.780 
O.79O 

I.042 

O.9OO 
I.O44 

O.9OO 

0.866 

0.808 

1.008 

0.900 
0.866 
0.772 

O.789 

O.687 
O.770 
O.73O 
O.681 
I.052 
I.032 

O.77O 
O.708 
O.681 

I. OI 7 

O.756 

Other  experiments  are  in  general  good  agreement  with  these.     The 
effect  of  work  in  the  fasting  animal  is  seen  almost  immediately.     In 


360 


PHYSIOLOGICAL    CHEMISTRY. 


the  last  experiments  the  respiratory  quotient  is  greater  than  unity. 
This  may  be  due  in  part  to  slight  errors  in  observation,  but  it  should 
be  remembered  that  there  are  classes  of  compounds  in  which  such  a 
result  would  always  follow.  Such  compounds  are  not  common  articles 
of  food,  but  often  make  a  part  of  certain  vegetable  foods.  The  com- 
plete oxidation  of  tartaric  acid,  for  example,  would  yield  a  quotient 
of  1.6. 

The  quotient  may  sometimes  be  high,  as  intimated  above,  if  the 
oxygen  has  been  at  first  absorbed  to  form  compounds  relatively  rich 
in  oxygen,  which  are  later  broken  down  rapidly,  under  working  or 
other  conditions.  If  an  observation  is  made  just  at  this  period  the 
excess  of  C02  liberated  would  present  an  abnormal  result.  For  char- 
acteristic results  the  observation  periods  should  be  as  long  as  possible. 

Illustrative  Case.  Some  idea  of  the  importance  of  the  respiratory 
coefficient  determination  may  be  obtained  from  a  consideration  of  the 
following  assumed  case  in  which  the  conditions  are  made  somewhat 
ideal  for  simplicity  of  calculation.  The  numerical  values  given  are 
such  as  might  be  obtained  from  the  mean  of  several  24-hour  experi- 
ments in  a  large  respiration  chamber.  The  diet  is  assumed  to  be  abun- 
dant and  the  tests  begun  after  a  condition  of  practical  nitrogen  equi- 
librium is  reached. 

Initial    weight 75  kilograms 

Final  weight  75-OS  kilograms 

Income  Observed. 


Wt.  in 
Grams. 

C 

Per  Cent. 

N 
Per  Cent. 

O 
Per  Cent. 

H 
Per  Cent. 

c 

Total. 

N 
Total. 

0 
Total. 

H 

Total. 

Proteins 

I50 

■     no 

44O 

35 
2,000 

53-5 

76.5 
44.2 

16.O 

23-5 
II.4 
49.6 

7.0 
12. 1 

6.2 

80.3 

84.2 

194-5 

24.O 

35-2 

12.5 

2l8.2 

IO.5 
13-3 

27-3 

Fats ... 

Carbohydrates... 
Salts 

Water 

Total 

2,735 

1 

359-o 

24.O 

265.9 

511 

Outgo  Observed. 


Weight  in  Grams. 

c. 

N. 

Salts. 

Vol.  co2. 

Respiration,  C02.. 
Respiration,  H20. . 
Urine,  H20 

932 
904 

i,35° 

74 

100 

33 

254.2 

8.7 

16.2 

20.4 
3-6 

3° 

'5 

471  1- 

Total  

3,393 

279.1 

24.0 

35 

The  weight  of  the  various  excreted  products  is  greatly  in  excess  of 


THE   GASEOUS   EXCRETION.  36 1 

the  visible  income,  but  the  oxygen  inhaled  has  not  yet  been  calculated. 
The  formula  given  above  may  be  applied  to  find  this : 

Oxygen  =      A'       -f-     Excreta   —         (A      +      F) 
75>050  3,393  75,ooo  2,735 

=  78,443  —  77,73s 

=  708  grams 
=  495.4  liters. 

In  the  table  above  the  volume  of  carbon  dioxide  eliminated  is  given  as 
471  liters.     The  respiratory  quotient  is  therefore 

495-4 
This  gives  us  the  first  clue  as  to  the  nature  of  the  foods  metabolized. 
The  factor  is  so  much  larger  than  that  corresponding  to  the  fats  that 
we  may  practically  exclude  these  at  once.  In  any  event  there  is  a  large 
protein  metabolism  since  the  original  nitrogen  of  the  food  is  all  found 
in  the  urine  and  the  feces.  In  other  words,  we  have  nitrogen  equi- 
librium, with  no  storing  up  of  protein  in  the  tissues.  The  respiratory 
quotient  corresponds  to  the  combustion  of  carbohydrates  and  proteins 
mixed. 

If  we  assume  for  the  moment  that  no  fat  is  oxidized,  this  calculation 
may  be  made.  The  254.2  gm.  of  carbon  in  the  carbon  dioxide  of 
respiration  calls  for  678  gm.  of  oxygen.  The  difference  between  this 
and  the  calculated  absorbed  oxygen,  708  gm.,  amounts  to  30  gm.,  which 
must  be  used  up  in  oxidizing  hydrogen  of  protein  substances.  This 
conclusion  is  drawn  because  the  carbohydrates  contain  enough  oxygen 
to  burn  their  own  hydrogen,  and  the  protein  nitrogen  appears  as  urea 
and  calls  for  no  outside  oxygen.  The  burning  of  fat  hydrogen  is 
excluded  in  the  assumption. 

The  nitrogen  of  the  feces  corresponds  to  22.5  gm.  of  original  pro- 
tein (6.25  X  3-6).  Not  all  of  this  nitrogen  is  actually  in  the  form  of 
unchanged  or  residue  protein ;  a  part  of  it  represents  products  of 
metabolism  which  are  excreted  in  the  feces,  as  explained  in  a  previous 
chapter.  Probably  a  considerable  fraction  may  be  considered  in  that 
form ;  but  it  must  be  counted  as  a  loss  to  the  body,  and  we  have  there- 
fore as  net  available  protein  (actually  used)  about  127.5  gm.  In  the 
final  metabolism  of  this  the  nitrogen  appears  in  urine  in  several  forms, 
but  mostly  as  urea.  The  per  cent  of  nitrogen  in  this  is  46.7.  In  some 
of  the  other  compounds  the  nitrogen  is  higher  and  in  some  lower.  In 
ammonia  much  hydrogen  (relatively)  is  held,  and  in  uric  acid  little. 
In  some  cases  there  is  an  excess  of  carbon  and  in  other  cases  relatively 
little  carbon  is  held  with  the  same  weight  of  nitrogen.     The  various 


362 


PHYSIOLOGICAL    CHEMISTRY. 


conditions  balance  each  other  pretty  well,  so  that  no  great  error  will 
be  made  if,  for  our  special  purpose,  we  count  all  the  excreted  nitrogen 
as  combined  in  the  form  of  urea.     We  have  then  these  relations : 


C. 

N. 

H. 

0. 

68.3 
8.7 

20.4 
20.4 

8.9 
2.9 

29.9 

In  urea 

11. 6 

59-6 

OO.O 

6.0 

18.3 

To  oxidize  this  remaining  carbon  requires  159.8  gm.  of  oxygen.  The 
18.3  gm.  of  protein  oxygen  will  oxidize  about  2.3  gm.  of  hydrogen. 
The  remaining  hydrogen  from  the  6  gm.  will  call  for  about  29.6  gm.  of 
oxygen,  which  corresponds  closely  -to  the  amount  calculated  above. 

The  ingested  carbon  is  seen  from  the  table  to  be  359  gm. ;  the 
excreted  carbon  is  279  gm.,  from  which  it  follows  that  the  body  has 
gained  80  gm.  If  we  assume  this  to  be  in  the  form  of  fat  the  latter 
must  amount  to  about  104  gm.  This  represents  the  true  gain  in  body 
weight ;  the  weighings  showed  a  gain  of  only  50  gm.  The  discrepancy 
may  be  accounted  for  by  assuming  an  excessive  excretion  of  urine. 
No  such  discrepancy  would  appear  if  the  urine  were  passed  from  the 
bladder  as  fast  as  formed,  but  as  it  is  collected  at  intervals  it  is  not 
possible  to  obtain  exactly  comparable  results. 

In  the  feces  there  must  be  some  carbon  derived  from  fats;  but  the 
amount  cannot  be  large,  because  for  the  nitrogen  of  the  feces  we  must 
calculate  at  least  10  or  12  gm.  to  correspond.  This  would  leave  about 
5  gm.  of  carbon  from  other  sources,  accounting  for  the  discrepancy 
between  consumed  fat  and  deposited  fat. 

The  above  calculations  illustrate  the  principles  involved;  in  an  actual  practical 
observation  the  method  would  be  the  same,  but  the  interpretation  of  results  might 
not  be  as  simple,  especially  with  a  low  respiratory  quotient  found.  In  the  above 
tables  the  salts  taken  with  the  food  are  assumed  to  include  those  to  be  formed  by 
the  oxidation  of  the  protein,  and  the  latter  substance  figured  as  income  is  assumed 
to  consist  of  the  organic  elements  only.  A  slight  error  is  introduced  in  the  calcu- 
lation in  this  way,  but  that  is  not  considered.  In  actual  practice,  of  the  carbo- 
hydrates some  little  would  escape  complete  metabolism.  The  above  results  would 
correspond  to  a  completely  burned  carbohydrate. 

In  the  above  table  of  observations  the  total  oxygen  in  the  consumed  substances, 
including  the  water,  is  2,044  gm-  In  the  excreted  products,  allowing  30  gm;  for  the 
solids  of  the  urine  and  feces  coming  from  bodies  other  than  the  original  salts,  the 
oxygen  appears  to  amount  to  about  2,800  gm.  The  difference  shows  an  excess  of 
756  gm.  while  the  calculation  above  gave  708  gm.  of  oxygen  taken  in.  The  dis- 
crepancy is  due  to  the  excess  of  water  excreted  as  urine.  It  will  be  noticed  also 
that  there  is  a  great  excess  of  excreted  water  over  the  2,000  gm.  consumed.  This 
amounts  to  over  350  gm.,  of  which  300  gm.  would  come  from  the  combustion  of  the 
carbohydrates  and  proteins  metabolized. 


THE    GASEOUS   EXCRETION.  363 


SKIN  RESPIRATION. 


It  is  usually  assumed  that  the  gaseous  exchange  is  wholly  through 
the  lungs,  but  this  is  not  quite  correct.  Experiments  with  men  and 
animals  have  shown  an  absorption  of  oxygen  and  an  escape  of  carbon 
dioxide  through  the  skin.  A  number  of  observers  have  put  results 
for  the  latter  on  record  which,  however,  are  not  in  good  agreement. 
For  1.6  square  meters  of  skin  surface  the  results  found  in  seven 
observations  varied  from  2.2  gm.  to  32  gm.  in  24  hours  The  last 
result  is  probably  much  too  high.  It  has  been  noticed  further  that  the 
amount  of  carbon  dioxide  escaping  through  the  skin  is  increased 
greatly  by  temperature.     The  excretion  at  30°  seems  to  be  several 


times  as  great  as  at  200. 


times  as  great  asawu  . 

For  the  absorption  of  oxygen  no  exact  figures  are  given,  but  the 
amount  is  very  small.  In  some  of  the  lower  animals,  however  a  large 
part  of  the  absorbed  oxygen,  as  well  as  of  the  excluded  carbon  dioxide, 
may  be  by  way  of  the  skin.  This  has  been  shown  especially  in  the 
frog,  where  after  removal  of  the  lungs  a  nearly  normal  exchange  may 
be  noted  for  a  period  of  days. 

The  question  of  the  excretion  of  other  gases  than  carbon  d.ox.de  by 
the  skin,  and  the  lungs  also,  has  been  much  discussed.     Formerly  it 
was  held  that  a  very  appreciable  quantity  of  organic  gaseous  bodies  is 
given  off  through  the  skin  and  this  elimination  was  considered  neces- 
sary for  the  well  being  of  the  body.     The  unpleasant  odor  of  the  a  r 
of  a  crowded  room  was  ascribed  to  these  organic  emanations.     But 
much  doubt  has  been  thrown  on  this  notion  by  various  «*«""* 
some  of  which  are  of  very  recent  date,  which  seem  to  show  that  these 
odors  come,  not  through  the  skin,  but  from  decaying  substances  on  the 
surface  of  the  skin  or  from  the  clothing,  if  it  is  old  and  soiled.     Ex- 
Z  ments  have  been  made  of  testing  the  air  drawn  through  a  small 
Ration  chamber,  enclosing  the  body  of  a  tnan  to  the  neck,  with 
perfectly  clean  skin  and  clothed  in  fresh,  clean  garments.     Such  air  is 
pr       c Jly  without  odor  and  has  no  action  on  solutions  of  perman 
Late  through  which  it  is  aspirated.     It  is  free  from  ammonia.     The 
ooors  of  perforation  are  apparently  largely  due  to  the  fermen  a  ion 
changes  of  solid  or  semi-solid  substances  on  the  surface  of  the  skin 
ra  her  than  to  excreted  gaseous  products  passing  through  the  pore 
vTh    he  water.     It  has  been  found  also  that  the  whole  surface  of  the 
Tody  may  be  covered  with  varnish  without  harmful  result  if  precau- 
tions  arc  taken  to  prevent  loss  oi  beat. 


364  PHYSIOLOGICAL    CHEMISTRY. 

TIME  AND  PLACE  OF  OXIDATION. 

The  determination  of  the  respiratory  quotient  through  short  inter- 
vals shows  considerable  variations,  as  pointed  out  some  pages  back. 
The  human  organism  has  not  the  power  of  storing  up  oxygen  in  the 
free  or  combined  form  through  a  long  period,  as  appears  to  be  the 
case  with  some  cold-blooded  animals,  which  are  able  to  exist  for  a  time 
in  an  atmosphere  free  from  oxygen.  With  man  and  warm-blooded 
animals  in  general  this  is  not  possible;  with  these  life  without  oxygen 
may  be  maintained  for  but  a  few  minutes  at  most.  An  exception 
exists  in  the  case  of  those  animals  which  pass  the  winter  in  a  dormant 
condition  (hibernating  animals)  and  for  human  beings  in  trance. 
Here  the  absorption  of  oxygen  and  excretion  of  carbon  dioxide  are 
reduced  to  a  minimum.  But  ordinarily  man  and  the  higher  animals 
require  some  inflow  of  oxygen  all  the  time. 

The  extent  to  which  this  oxygen  is  used  depends  on  the  activity  of 
the  muscles  largely.  In  rest  periods  the  amount  of  oxygen  taken  up 
by  the  muscles  is  much  greater  than  is  the  carbon  dioxide  given  off, 
but  with  the  contracting  or  working  muscle  the  reverse  is  the  case.  In 
experiments  in  which  the  changes  in  the  blood  supply  of  individual 
muscles  may  be  followed  it  may  be  shown  that  for  rest  periods  the 
respiratory  quotient  for  the  muscle  may  fall  far  below  0.7  or  even 
below  0.5.  From  the  rapidly  contracting  muscle,  on  the  other  hand, 
the  evolution  of  carbon  dioxide  is  relatively  great.  A  respiratory 
quotient,  for  the  muscle,  of  1.5  or  even  2  or  more  may  be  found.  This 
indicates  that  during  rest  oxygen  may  be  taken  up  from  the  blood  and 
held  or  condensed  in  some  manner  by  substances  within  the  muscular 
tissue,  but  of  the  mechanism  of  this  reaction  unfortunately  but  little  is 
known.  In  doing  work  tissue  is  rapidly  oxidized  at  the  expense  of  the 
stored-up  oxygen,  and  a  great  excess  of  carbon  dioxide  is  given  off 
quickly.  These  changes  follow  one  upon  the  other  rather  rapidly.  In 
the  oxygen-absorbing  stage  some  intermediate  products  are  probably 
built  up  from  sugar  or  glycogen  or  other  substances,  which  fall  apart 
with  liberation  of  water  and  carbon  dioxide  in  the  succeeding  active 
condition  of  the  muscle. 

The  problem  of  oxidation  in  the  tissues  is  possibly  somewhat  like 
that  of  etherification  in  which  alcohol  yields  ether  indirectly  through 
the  intermediate  ethyl  sulphuric  acid,  and  several  suggestions  have 
been  brought  forward  as  to  the  character  of  complexes  formed  in  one 
stage  of  the  oxidative  metabolism  to  be  decomposed  in  another.  At 
the  present  time  these  suggestions  are  practically  wholly  within  the 
realm  of  speculation,  and  not  therefore  suitable  for  presentation  in  this 


THE   GASEOUS   EXCRETION.  365 

place.  It  is  likely  that  all  these  reactions,  which  seem  to  be  carried  on 
in  the  tissues  rather  than  in  the  fluids  of  the  body,  are  incited  by 
enzymic  ferments,  and  to-day  certain  classes  of  oxidases  are  often 
assumed  to  be  the  agents  active  in  the  changes.  For  some  of  the 
oxidations  it  has  been  pretty  well  settled  that  an  enzyme  produced  by 
the  pancreas  is  necessary. 


CHAPTER   XXII. 

THE  ENERGY  EQUATION. 

We  come  now  to  a  brief  consideration  of  one  of  the  most  important 
questions  connected  with  the  whole  animal  chemistry,  and  this  is  the 
question  of  the  liberation  of  energy  from  the  consumption  of  various 
foods.  The  function  of  the  food  we  eat  is  a  multiple  one.  It  may  not 
only  increase  the  body  weight  and  maintain  the  various  functions  of 
the  body  through  oxidation,  but  in  its  combustion  heat  is  liberated  to 
maintain  also  the  body  temperature,  and  energy  is  furnished  to  enable 
us  to  perform  external  work.  It  is  interesting  to  measure  the  effect 
of  the  food  in  these  several  directions,  which  may  be  done  with  a  fair 
degree  of  accuracy.  The  following  considerations  will  show  the  basis 
of  the  calculations. 

POTENTIAL  ENERGY  OF  FOOD. 

The  food,  consisting  essentially  of  combustible  substances,  is  the 
source  of  a  large  amount  of  potential  energy.  In  the  complete  com- 
bustion of  the  fats,  carbohydrates  and  proteins  of  the  food  a  large 
amount  of  heat  is  liberated  and  this  in  turn  is  the  equivalent  of  a 
certain  amount  of  work.  The  potential  energy  of  chemical  substances 
may  be  measured  in  various  ways,  but  for  purposes  like  the  present  it 
is  customary  to  measure  this  energy  in  terms  of  the  units  of  heat  lib- 
erated in  the  combustion  of  the  body  in  question  with  oxygen.  Cer- 
tain units  are  in  common  use : 

Unit  of  Heat,  Calorie.  The  unit  of  heat  or  calorie  may  be  defined 
as  the  quantity  of  heat  required  to  raise  the  temperature  of  a  gram  of 
water  one  centigrade  degree,  at  a  mean  temperature.  As  the  heat 
absorption  of  a  gram  of  water  is  not  quite  the  same  throughout  the 
scale,  the  calorie  is  perhaps  more  satisfactorily  defined  as  the  one  hun- 
dredth part  of  the  quantity  of  heat  required  to  raise  the  temperature 
of  a  gram  of  water  from  o°  to  ioo°  C.  This  gives  the  ordinary,  or 
small  calorie.  In  dealing  with  large  heat  transfers  a  larger  unit  is 
preferable  and  one  just  1,000  times  as  large  is  frequently  used.  In 
this  the  kilogram  in  place  of  the  gram  of  water  is  warmed,  and  the 
unit  is  called  the  large  calorie.  The  first  may  be  abbreviated  cal.  and 
the  second  Cal. 

366 


THE    ENERGY    EQUATION.  367 

Unit  of  Work  and  Unit  of  Force.  The  unit  of  force  is  called  the 
dyne  and  may  be  defined  as  the  force  which,  acting  for  I  second  on  a 
mass  of  1  gram,  gives  to  it  an  acceleration  of  I  centimeter  per  second. 
The  force  of  gravity  at  the  sea  level  is  about  981  dynes,  since  this  adds 
to  a  falling  body  an  acceleration  of  981  cm.  per  second. 

The  unit  of  work  is  the  erg,  and  it  may  be  defined  as  the  work  done 
in  overcoming  unit  force  through  unit  distance.  One  dyne  acting 
through  one  centimeter  gives  us  one  erg  of  work.  To  lift  1  gram 
through  1  centimeter  requires  981  ergs  of  work. 

Mechanical  Equivalent  of  Heat.  Work  may  be  done  by  the  proper 
utilization  of  heat,  and  in  turn  work  may  be  wholly  converted  into 
heat.  It  is  possible,  therefore,  to  express  one  in  terms  of  the  other. 
The  mechanical  or  work  equivalent  of  a  unit  of  heat  has  been  deter- 
mined many  times  by  very  elaborate  experiments.  If  a  given  quantity 
of  heat  could  be  applied  wholly  to  the  lifting  of  a  weight  it  would  be 
found,  in  accordance  with  the  mean  results  of  these  experiments,  that 
1  calorie  would  be  able  to  lift  423.5  gm.  through  1  meter,  or  1  gm.  of 
substance  through  423.5  meters.  Conversely,  if  a  gram  of  water  be 
dropped  from  a  height  of  423.5  meters,  and  its  energy  of  motion  wholly 
converted  into  heat,  its  temperature  will  be  found  to  be  increased  i° 
C.     We  have  then  these  relations : 

1  calorie  =  42,350  gm.  cm. 
=  41,500,000  ergs. 

Heats  of  Combustion.  By  means  of  calorimeter  experiments  the 
following  heats  of  combustion  have  been  determined.  Results  found 
by  different  workers  show  slight  variations,  but  the  values  here  are 
mean  values  and  sufficient  for  illustration.  The  number  of  calories 
furnished  by  burning  1  gm.  of  substance  in  each  case  is  given. 

Table  of  Heats  of  Combustion. 

Hydrogen    34,200  Cane  sugar  4,000 

Carbon    8,100  Starch     4,200 

Ethyl  alcohol   7,060  Casein   5,700 

Glycerol    4,200  Egg  albumin    5,7oo 

Mannitol    4,000  Urea    2,500 

Palmitic  acid   9,300  Uric  acid  2,700 

Stearic  acid    9,400  Leucine    6,500 

Fats,  average   9,400  Tyrosine    6,000 

Hexoses    3,700  Creatine,  anhyd  4,250 

These  values  are  for  complete  combustion,  but  as  the  proteins  in  the 
body  are  not  oxidized  to  leave  water,  carbon  dioxide  and  nitrogen,  we 
must  subtract  from  the  given  values  the  heats  of  combustion  of  the 


Potential  energy  of 
Food. 


368  PHYSIOLOGICAL    CHEMISTRY. 

urea,  uric  acid,  creatinine  and  other  products  found  in  the  urine,  in 
order  to  secure  the  physiological  heats  of  combustion,  with  which  we 
are  practically  concerned. 

DISTRIBUTION  OF  FOOD  ENERGY. 

With  these  preliminary  considerations  we  are  able  to  look  at  the 
manner  in  which  the  energy  of  the  consumed  food  is  distributed.  On 
the  one  side  we  have  the  substance  burned,  on  the  other  the  products, 
which  may  be  represented  diagrammatically  in  this  way : 

Potential  energy  of 

Flesh  gained. 
Feces. 
Urine. 

Perspiration 
Kinetic  energy  of 

Work. 
Heat. 

Experimentally,  the  whole  of  the  kinetic  energy  may  be  made  to  take 
the  form  of  heat,  which  simplifies  the  observations  materially.  It  is 
practically  possible  to  determine  the  heat  liberation  in  the  large  respi- 
ration calorimeters  already  referred  to,  and  the  use  of  such  apparatus 
will  be  explained  below.  First,  however,  a  general  method  of  calcu- 
lating the  energy  liberated  as  heat  will  be  given. 

CALCULATION  OF  KINETIC  ENERGY  OF  FOOD. 

In  illustration  of  this  we  may  make  use  of  the  example  given  in  the 
last  chapter,  and  employ  a  method  which  in  principle  is  very  simple. 
The  income  of  energy  is  due  to  the  consumption  of  certain  weights  of 
protein,  fat  and  carbohydrates,  the  last  of  which  we  may  assume  is 
made  up  of  9  parts  of  starch  and  1  part  of  cane  sugar,  all  weights 
referring  to  the  anhydrous  condition.  The  effect  of  the  oxidation  of 
sulphur  and  phosphorus  will  be  neglected  here,  and  the  protein  will  be 
assumed  pure  carbon,  hydrogen,  oxygen  and  nitrogen.  We  have  then 
as  income : 

From  150  gm.  protein,  150X5700=    855,000 

no  gm.  fat,  110X9400=1,034,000 

440  gm.  carbohydrate,  440X4180  =  1,839,200 

3,728,200 

In  small  calories  the  whole  income  is  therefore  equivalent  to  3,278,- 
200  cal. 

We  have  next  to  calculate  the  potential  energy  of  the  food  stuffs 


THE    ENERGY    EQUATION.  369 

not  actually  consumed,  which  are  left  in  the  feces  and  the  urine,  and 
also  the  energy  of  any  substance  which  may  be  put  down  as  a  gain  in 
weight  in  the  body.  Recalling  the  data  of  the  experiment  in  the  last 
chapter  we  have 

l33  HP*1-  feces  with  16.2  gm.  C. 
1,424  gm.  urine  with  20.4  gm.  N. 

Calculating  the  N  of  the  urine  as  urea,  which  in  practice  would  not  be 
quite  accurate,  we  have  44  gm.  of  t.hat  substance.  The  organic  matter 
of  the  feces  corresponds  approximately  to  22.5  gm.  of  bodies  resem- 
bling protein  and  5.5  gm.  of  bodies  resembling  fats,  and  these  data  we 
can  now  employ  in  the  calculation. 

The  illustration  gave  also  a  gain  of  80  gm.  of  fat.  The  solid  matter 
lost  in  the  form  of  perspiration  is  so  small  that  it  may  be  ignored  for 
the  present  purpose.     We  have  then  the  following  deductions  to  make : 

Potential  energy  in     80  gm.  of  fat  stored    752,000 

!33  gm-  of  feces 180,000 

1,424  gm.  of  urine    1 10,000 

1,042,000 
This  leaves  as  a  balance  to  be  calculated  as  kinetic  energy 

3,728,200 
1,042,000 

2,686,200  calories 

Another  method  of  calculation  deals  with  the  carbon  and  hydrogen  of  the  food 
and  feces  only.  The  heat  production  was  at  one  time  assumed  to  depend  on  the 
combustion  of  the  carbon  of  the  fats,  carbohydrates  and  proteins  and  the  hydrogen 
of  the  fats  and  proteins.  The  hydrogen  of  the  carbohydrates  was  not  considered 
because  it  was  supposed  to  be  closely  combined  with  the  oxygen  present  in  the  same 
compounds  in  such  a  form  as  to  yield  no  more  heat  on  oxidation.  In  like  manner 
the  combustion  heats  of  the  urine  and  feces  may  be  calculated  from  the  whole 
carbon  and  hydrogen  content  of  the  organic  substances.  The  total  carbon  of  the 
food  in  the  experiment  is  395  gm.,  of  the  hydrogen  in  fats  and  proteins  23.8  gm. 
The  carbon  of  the  urine  and  feces  is  24.9  gm.,  while  the  hydrogen  of  the  urine  and 
feces  is  about  5.2  gm.     We  have  then : 

Heat  units  from  359  gm.  of  food  carbon 2,907,900 

Heat  units  from  23.8  gm.  of  food  hydrogen. . ..      813,900 

3.721,800    3,721,800 

Heat  units  from  24.9  gm.  of  excreta  carbon 201,600 

Heat  units  from    5.2  gm.  of  excreta  hydrogen. .      177.840 

379-530       379,530 

Net  calories  3,342,270 

From  this  result  the  value  of  the  energy  stored  as  fat  would  have  to  be  subtracted 
as  before.  This  method  of  calculation  gives  a  somewhat  lower  result  than  the  other, 
and  largely  because  of  the  uncertainty  in  allowing  for  the  excreted  carbon  and 
hydrogen,  but  it  has  value  as  a  comparison  process. 

25 


370  PHYSIOLOGICAL    CHEMISTRY. 

Respiration  Calorimeters.  In  experiments  with  men  or  large  animals  on  the 
combustion  of  food  and  liberation  of  heat  some  kind  of  respiration  apparatus  is 
employed.  Some  modification  of  a  type  originally  introduced  by  Pettenkofer  is 
generally  used.  In  this  the  subject  is  placed  in  a  chamber  with  double  walls  through 
which  a  current  of  air  may  be  forced  and  uniformly  mixed  inside.  A  known  part 
of  the  ingoing  air  and  of  the  outgoing  air  may  be  diverted  for  analysis  so  as  to 
permit  an  exact  determination  of  the  amount  of  oxidation  products  liberated  at  any 
time.  The  Atwater  and  Rosa  calorimeter  is  the  most  complete  of  all  such  con- 
structions. In  this  the  heat  liberated  by  the  subject  is  taken  up  by  a  current  of 
cold  water  circulating  through  numerous  toils  of  pipe  inside  the  chamber  and  in 
such  a  way  as  to  maintain  a  perfectly  uniform  temperature  in  the  chamber  space. 
The  walls  of  the  chamber  are  made  of  compartments  containing  two  layers  of 
air  and  two  layers  of  water  maintained  in  such  relations  that  they  prevent  gain  or 
loss  of  heat.  The  whole  heat  liberation  is  taken  up  by  the  circulating  water  and 
may  be  accurately  measured.  The  respiration  chamber  has  a  capacity  of  about 
175  cubic  feet  and  is  large  enough  to  contain  a  chair  and  small  table  for  the  con- 
venience of  the  occupant  and  a  couch  to  sleep  on  at  night.  The  construction  is 
such  that  food  may  be  passed  in  and  the  urine  and  feces  removed  without  making 
any  appreciable  change  in  the  temperature  or  content  of  the  air  inside.  With  such 
an  apparatus  it  is  possible  to  carry  on  a  test  of  many  days  duration  and  obtain 
extremely  accurate  and  important  results. 

In  work  experiments  in  such  a  calorimeter  a  bicycle  is  mounted  so  that  work  is 
done  against  friction.  The  final  effect  is  increased  heat  liberation,  measured  as 
before. 

The  construction  of  this  large  calorimeter  suggested  the  building  of  still  larger 
ones  of  the  same  general  type.  Some  of  these  are  being  used  in  agricultural  experi- 
ment stations  in  metabolism  experiments  on  large  animals,  from  which  results  of 
great  practical  value  may  be  expected. 

DISTRIBUTION  OF  THE  HEAT  ENERGY. 

According  to  the  first  calculation  above  we  have  from  the  700  grams 
of  food  consumed  a  balance  of  2,686,200  calories.  It  remains  to  show- 
about  how  this  may  be  dissipated.  If  retained  in  the  body  it  would 
soon  bring  the  latter  to  the  boiling  point.  But  the  heat  liberated  in 
the  combustion  of  the  food  finds  several  outlets,  the  most  important  of 
which  will  be  now  indicated.  In  the  first  place  the  urine  and  feces 
leave  the  body  at  a  temperature  much  higher  than  that  of  the  water 
consumed;  the  water  of  respiration  and  perspiration  has  to  be  vapor- 
ized at  the  expense  of  heat ;  the  air  inhaled  is  warmed  to  a  temperature 
of  370,  which  is  in  the  mean  20 °  higher  than  when  taken  in.  The 
specific  heat  of  the  air  (at  constant  pressure)  is  about  0.25.  We  have 
then,  approximately,  the  following  relations,  assuming  15  kilograms 
of  air  to  be  inhaled  in  the  24  hours : 

To  warm  15,000  gm.  air  200 7S,ooo  cal. 

To  warm  1,557  &m-  urine  and  feces  200  31,140 

To  evaporate  904  gm.  of  water  (904  X  580)   524,320 

630,460 


THE    ENERGY    EQUATION.  371 

This  number  of  calories  must  be  taken  from  the  net  produced  calories 
to  obtain  the  heat  radiated  or  otherwise  lost  by  the  body.  We  have 
then  this  difference : 

2,686,200 
630,460 

2,055,740 
That  is,  something  over  2,000,000  calories  are  dissipated  by  radiation. 

HEAT  RADIATION  WHEN  WORK  IS  DONE. 

All  these  calculations  are  based  on  the  assumption  that  no  mechan- 
ical work  is  being  done  by  the  person  under  observation,  or  if  done  it 
is  finally  all  converted  into  heat.  In  experiments  in  the  respiration 
calorimeter  a  very  close  agreement  is  found  between  the  calculated 
and  observed  heat  or  energy  liberations.  This  is  illustrated  by  the 
results  of  one  of  the  Atwater  and  Rosa  experiments.  The  figures  are 
the  daily  means  from  tests  running  through  4  days : 

Total  energy  of  food,  determined 3,6/8  Cal. 

Energy  of  urine  and  feces  264 

Net    energy    3,4T4 

Energy  of  fat  lost  488 

3,002 
Energy  stored  as  protein  38 

Total  energy  of  material  actually  oxidized 3,864 

Heat  actually  measured    3,739 

125 

There  is,  therefore,  a  difference  of  only  125  large  calories  in  this  test, 
which  was  one  of  the  early  ones  with  the  new  apparatus.  In  later 
experiments  described  by  Atwater  and  his  colleagues  much  closer 
results  have  been  reported,  which  shows  the  general  correctness  of  the 
method  of  calculation  followed. 

Effect  of  Work.  To  maintain  the  individual  at  work  a  greater 
expenditure  of  energy  is  necessary,  and  the  total  energy  of  substances 
metabolized  must  be  balanced  by  the  heat  liberated  and  external  work 
done,  fn  this  connection  it  may  be  well  to  recall  some  relations  first 
pointed  out  by  flirn,  in  which  ;i  comparison  is  drawn  between  the 
work  of  man  and  an  engine.  In  both  cases  the  work  is  accomplished 
through  the  expenditure  of  the  potential  energy  stored  up  in  carbo- 
naceon-  substances,  food  in  the  one  instance,  coal  in  the-  other.  As  the 
illustrations  above  show,  the  heat  from  the  food  is  practically  constant, 
whether  it  be  evolved  through  oxidation  in  the  body  or  in  a  calorimeter. 


372  PHYSIOLOGICAL    CHEMISTRY. 

Imagine  now  a  small  steam  engine  burning  a  constant  amount  of  coal 
inside  a  calorimeter.  In  one  case  let  no  work  be  done  by  the  piston; 
the  heat  of  the  steam  is  not  employed  in  expansion,  but  is  totally 
absorbed  by  the  water  of  the  calorimeter,  which  takes  up  a  certain 
number  of  calories  that  may  be  accurately  noted.  In  a  second  case 
allow  the  same  amount  of  coal  to  be  burned  under  the  boiler  of  the 
small  engine  in  the  same  time,  but  let  the  engine  do  work  outside  the 
calorimeter,  which  may  be  accomplished,  for  example,  by  means  of  a 
small  shaft  passing  through  the  walls  of  the  calorimeter  in  such  a  way 
as  to  convey  no  appreciable  amount  of  heat.  It  will  now  be  found  that 
the  gain  in  temperature  in  the  water  of  the  calorimeter  is-  less  than 
before  for  the  same  coal  consumption,  and  that  the  difference  is  meas- 
ured by  the  external  work  alone.  One  calorie  less  in  the  calorimeter 
heat  corresponds  to  423.5  gram-meters  of.  work  done  through  the 
agency  of  the  shaft. 

With  an  animal  the  case  is  different.  The  doing  of  external  work 
necessitates  always  the  burning  of  more  food  than  is  the  case  with  the 
fasting  metabolism,  when  the  energy  requirement  is  for  doing  internal 
work,  as  will  be  explained  below.  In  the  engine  the  amount  of  coal 
burned  may  be  constant  whether  work  is  done  or  not.  However,  with 
the  animal  this  result  is  noticed :  Increased  food  consumption,  with 
increased  oxidation,  may  not  be  accompanied  by  an  increase  in  work; 
in  this  case  there  must  be  an  increased  liberation  of  heat.  If  work  is 
done,  there  is  still  an  increase  in  the  liberation  of  heat,  but,  as  with  the 
machine,  we  must  subtract  the  heat  equivalent  of  the  accomplished 
work.  A  part  of  the  increased  heat  liberation  is  called  for  by  increased 
internal  work  also.  It  follows,  therefore,  that  the  working  animal  is 
warmer  than  the  passive  animal,  but  the  increase  is  not  proportional 
to  the  food  consumed  or  oxygen  absorbed. 

External  Work  Equivalent.  Although  the  animal  is  able  to  con- 
vert but  a  limited  portion  of  the  potential  energy  of  the  food  into 
external  work,  as  a  machine  it  is  still  much  more  perfect  than  the  steam 
engine.  This  is  especially  true  of  man.  In  the  best  steam  engines 
not  more  than  about  12  per  cent  of  the  potential  energy  of  the  fuel 
can  be  recovered  in  the  form  of  work.  In  animals,  through  a  short 
period,  the  transformation  may  amount  to  as  much  as  35  per  cent 
of  the  net  available  potential  energy.  In  making  such  comparisons, 
however,  it  must  be  remembered  that  the  animal  can  work  but  a  limited 
time.  In  the  rest  periods  of  the  animal  the  loss  of  heat,  without  any 
corresponding  mechanical  gain,  goes  on. 


THE    ENERGY    EQUATION.  373 

The  law  defining  the  maximum  conversion  of  heat  into  work  through  the  steam 
engine  is  well  known.  The  extent  of  the  limitations  in  the  animal  are  not  known. 
In  the  steam  engine  the  limitation  depends  on  the  relation  of  the  highest  heat  of  the 
steam  to  the  temperature  of  the  condenser.  If  T  is  the  absolute  temperature  of 
the  live  steam  and  t  the  temperature  of  the  condenser  the  maximum  transforma- 
tion of  heat  into  work  cannot  be  greater  than 

T  —  t 


THE  INTERNAL  WORK  OF  THE  ANIMAL. 

Even  when  the  animal  appears  passive  a  great  deal  of  work  is  going 
on  which  may  be  described  as  internal  work.  The  nature  and  extent 
of  some  of  this  is  known  with  a  fair  degree  of  accuracy,  while  for  the 
extent  of  the  metabolism  corresponding  to  other  kinds  of  work  we 
have  not  much  beyond  conjecture.  It  is  possible  to  calculate  approxi- 
mately the  work  done  in  maintaining  the  circulation  of  the  blood,  and 
in  respiration,  and  some  attempts  have  been  made  to  estimate  the  work 
of  the  other  muscles  at  rest ;  but  to  approximate  the  work  done  in  mas- 
ticating, digesting,  transporting  and  transforming  the  food  stuffs  is 
much  more  difficult.  The  work  of  the  heart  alone  has  been  estimated 
at  from  20,000  to  60,000  kilogram-meters  in  24  hours.  Three  thou- 
sand Cal.  of  heat  liberated  would  correspond  to  1,270,500  kilogram- 
meters  of  work;  hence  the  heart  work  in  forcing  the  blood  through  the 
vessels  may  amount  to  as  much  as  5  per  cent  of  the  whole  metabolism. 

In  respiration  the  work  done  is  largely  the  expansion  of  the  thorax 
against  the  atmospheric  pressure  and  the  elastic  tension  of  the  rib 
cartilages  and  the  lungs.  The  conditions  for  estimation  are  perhaps 
more  favorable  than  in  the  other  case.  It  has  been  calculated  that  4 
to  5  per  cent  of  the  whole  metabolism  is  called  for  by  this  work. 

In  maintaining  the  tonus  of  the  great  mass  of  skeletal  muscles  of 
the  body  it  is  likely  that  a  large  metabolism  is  required.  The  muscular 
part  of  the  body  is  not  far  from  10  per  cent  in  the  mean,  or  40  per 
cent  of  dry  substance.  In  the  body  of  a  man  weighing  75  kilograms 
we  have  therefore  about  7.5  kilograms  of  muscle  substance.  A  large 
fraction  of  this  falls  to  the  so-called  skeletal  portion  which  exists 
always  in  a  peculiar  tense  condition.  In  keeping  up  this  condition 
without  doing  outside  work  oxidation  is  necessary.  Glycogen  is  split 
up  and  water  and  carbon  dioxide  appear.  The  only  visible  effect  of 
this  metabolism  is  the  production  of  heat.  When  the  muscle  docs 
outside  work,  as  in  lifting  a  weight,  although  the  heat  production  may 
be  greater  in  the  sum,  it  is  relatively  less  in  proportion  to  the  oxygen 
consumption.      A  pari  of  the  energy  is  consumed  in  lifting  the  weight. 


374  PHYSIOLOGICAL    CHEMISTRY. 

Heat  Production  Incidental.  It  is  possible  that  the  whole  heat 
liberation,  at  times,  is  but  a  result  of  the  various  kinds  of  internal  work 
done,  and  that  no  oxidation  takes  place  for  the  simple  production  of 
heat.  This  may  be  the  case  at  relatively  high  temperatures.  At  cer- 
tain lower  temperatures,  on  the  other  hand,  it  is  apparent  that  a  part 
of  the  heat  liberation  is  called  for  independently  of  that  transformed 
in  the  internal  work.  A  certain  heat  production  is  necessarily  con- 
nected with  the  performance  of  the  various  body  functions  and  this 
down  to  some  particular  external  temperature  limit  is  sufficient  for  the 
heat  demands  of  the  body.  Numerous  experiments  have  shown  that 
for  each  animal  species  there  is  an  external  temperature  at  which  the 
general  metabolism,  as  indicated  by  carbon  dioxide  excretion  or  oxygen 
consumption,  and  the  resultant  heat  liberation,  reach  a  minimum  value. 
This  temperature  limit  has  been  called  the  critical  temperature;  below 
it  the  metabolism  and  heat  liberation  increase,  evidently  not  in  conse- 
quence of  a  call  for  more  work  but  because  of  the  necessity  for  more 

heat. 

ISODYNAMIC  RATIOS. 

In  metabolism,  fats,  carbohydrates  and  proteins  all  yield  kinetic 
energy  and  to  a  large  extent  each  one  is  capable  of  replacing  the 
others,  a  certain  minimum  of  protein  being  always,  of  course,  neces- 
sarily present.  The  proportions  in  which  they  may  replace  each  other 
may  be  found  by  a  variety  of  experimental  methods,  which  yield  fairly 
concordant  results.  The  foods  may  be  burned  in  a  calorimeter  and  the 
heats  of  combustion  noted,  or  their  values  may  be  compared  through 
the  amounts  of  oxygen  required  by  calculation  to  oxidize  them,  or 
finally  animal  experiments  in  the  respiration  calorimeter  may  be  re- 
sorted to  to  fix  the  relative  values.  The  isodynamic  relations  are 
given  in  this  table  calculated  from  results  of  animal  experiments. 

ioo  gm.  of  fat  = 

Lean  meat,  dry   243  gm. 

Cane  sugar   234  gm. 

Glucose     256  gm. 

Starch   232  gm. 

For  such  substances  the  calculated  and  observed  values,  or  the  com- 
bustion calorimeter  and  the  respiration  calorimeter,  give  closely  agree- 
ing results,  but  it  must  be  remembered  that  many  compounds  which 
show  considerable  value  as  measured  by  combustion  are  absolutely 
worthless  as  measured  by  nutrition.  Creatinine  and  urea  are  illus- 
trations ;  both  are  products  of  metabolism.  On  the  other  hand,  alcohol 
shows  about  the  same  value  in  the  respiration  calorimeter  as  it  shows 


THE    EXERGY    EQUATION.  375 

in  the  combustion  calorimeter,  and  its  metabolic  value  would  therefore 
appear  high.  However,  the  actual  food  value  of  alcohol  is  practically 
low  because  limited  by  its  toxic  action. 

FOOD  CONSUMPTION  IN  SEVERE  MUSCULAR  EXERTION. 

In  the  last  chapter  reference  was  made  to  earlier  discussions  on  the 
question  of  the  kind  of  food  which  must  be  metabolized  to  enable  the 
animal  organism  to  do  work.  By  many  authorities  heat  liberation  was 
looked  upon  as  an  end  in  itself,  and  hence  foods  were  divided  into  the 
two  classes  :  those  important  in  the  production  of  heat  and  those  impor- 
tant in  the  production  of  outside  work.  The  fats  and  carbohydrates 
are  found  in  the  first  group  and  the  proteins  in  the  second.  One  of 
the  earliest  experimental  investigations  on  the  subject  was  the  classic 
one  of  Fick  and  Wislicenus,  already  referred  to.  These  two  men  in 
1866  made  the  ascent  of  the  Faulhorn  in  the  Swiss  Alps,  from  a 
known  level,  and  determined  the  excretion  of  nitrogen,  as  urea,  during 
and  following  the  ascent.  As  the  elevation  to  which  they  ascended 
was  known,  it  was  possible  to  make  some  calculations,  approximately 
accurate,  of  the  work  done  in  the  ascent.  Two  things  were  shown 
especially  by  the  tests  and  calculations :  there  was  not  a  great  increase 
in  the  urea  excreted,  and  secondly  the  protein  metabolized,  as  indicated 
by  the  urea  measured,  was  not  at  all  sufficient  to  account  for  the  work 
done.  Their  own  conclusions  were  that  the  protein  consumed  would 
not  furnish  more  than  half  or  three- fourths  the  energy  necessary  to 
lift  their  bodies  through  the  1956  meters  of  ascent,  to  say  nothing  of 
the  work  done  in  the  horizontal  direction  on  a  winding  pathway,  or 
of  the  internal  work  of  the  body.  This  experiment  attracted  a  great 
deal  of  attention.  Frankland  made  a  new  determination  of  the  heat 
of  combustion  of  protein  and  showed  that  the  value  assumed  by  Fick 
and  Wislicenus  was  far  too  high,  thus  making  the  discrepancy  still 
greater. 

Since  then  many  similar  observations  have  been  made  which  show 
pretty  clearly  that  when  fats  or  carbohydrates  are  abundant  in  the  food 
there  is  no  excessive  destruction  of  protein  in  the  performance  of  ordi- 
nary work.  With  the  ingestion  of  a  small  amount  of  protein  it  is  easy 
to  cover  the  normal  metabolism.  But  the  case  is  different  when  the 
work  is  hard.  Here,  even  with  abundant  food  and  plenty  of  protein, 
there  appears  to  be  some  loss  of  nitrogen  by  the  body.  In  other  words, 
more  tissue  is  broken  flown  than  is  formed  new.  This  is  brought  out 
clearly  in  the  observations  of  Atwater  on  the  work  done  by  bicyclers 
in  a   six-day  race  some  year-,  ago,   in  which  the    food  consumed  and 


376 


PHYSIOLOGICAL    CHEMISTRY. 


nitrogen  eliminated  were  carefully  watched.  The  following  table 
gives  a  summary  of  the  most  important  observations,  with  the  energy 
in  large  calories : 


Average 

Miles 

per 

Day. 

Protein  Daily. 

Energy  Daily. 

Rider. 

In  Total 
Food. 
Grams. 

In  Available 

Food. 

Grams. 

Metabolized. 
Grams. 

In  Total 
Food. 
Lai. 

In  Available 
Food. 
Cal. 

Metabolized. 
Cal. 

A 
B 
C 

334-6 
3°3 -8 

287.7 

169 
179 
211 

158 
163 
197 

223 
223 
243 

4,957 
6,300 
4,898 

4,547 
5,871 
4,323 

4,789 
6,066 
4,464 

Riders  A  and  B  rode  through  six  days.  C  rode  three  days.  The 
energy  metabolized  does  not  include  that  from  body  fat,  which  may 
have  been  considerable. 

DIETARIES. 

The  question  of  the  proper  amount  of  food  and  the  character  of  the 
food  for  different  kinds  of  work  has  been  very  thoroughly  studied  in 
the  past  few  years  and  a  large  number  of  observations  on  individuals, 
families  and  communities  of  soldiers,  prisoners  and  paupers  have  been 
collected.  The  diet  in  some  cases  is  known  to  be  sufficient,  in  others 
insufficient.  From  present  experience  it  is  possible  to  say  of  many 
dietaries  that  they  are  excessive,  and  probably  objectionable  in  conse- 
quence. The  following  table  illustrates  the  dietaries  of  a  great  many 
people  living  under  different  conditions.  The  figures  are  taken  mainly 
from  the  compilations  of  Konig  and  Atwater: 


Occupation,  Etc. 


Italian  laborers,  Chicago 

Bohemian  laborers,   Chicago 

Russian  laborers,   Chicago 

Laborers,  crowded  district,  New  York. 

Laborers,  low  income,  Pittsburgh 

Mechanics,  eastern  and  central  U.   S. . . 

French   Canadians,  Chicago   

French   Canadians,   Massachusetts    

American  professional  men    

Bavarian  workmen,  high  class 

Munich   prisons,   work 

Munich  prisons,  no  work  

Bavarian  soldiers,  war  

Bavarian  soldiers,  garrison  


(J? 

t  > 

Protein, 

Fat, 

Carbohy- 

.a  a 

Grams. 

Grams. 

0.2 

4 

I03 

Ill 

391 

8 

115 

103 

36o 

9 

137 

102 

4l8 

19 

106 

117 

367 

2 

8l 

97 

311 

14 

103 

150 

402 

5 

Il8 

158 

345 

5 

III 

193 

485 

14 

I05 

124 

420 

3 

151 

54 

479 

104 

38 

521 

87 

22 

305 

145 

100 

500 

120 

56 

500 

Calories. 


3,060 
2,885 
3,232 
3,030 
2,5IO 
3,465 
3,365 
4,235 

3,335 
3,o85 
2,916 
1,819 
3,575 
3,063 


The  above  results  are  fairly  representative  and  show  that  in  general 
over  3,000  Cal.  per  day  must  be  provided  in  the  food.  In  the  figures 
the  available  or  net  calories  are  calculated  from  1  gm.  protein  or 
carbohydrate  =  4. 1   Cal.,  1  gm.  fat  =  9.3  Cal.      But  many  extreme 


THE    ENERGY   EQUATION.  377 

results  are  also  found  in  the  literature.  For  prisoners  confined  in 
cells  and  not  working,  for  paupers  in  asylums,  and  even  with  laborers 
poorly  paid,  the  foods  consumed  may  not  yield  1,500  Cal.  On  the 
other  hand,  workmen  in  the  American  winter  lumber  camps,  who  as  a 
rule  are  well  paid,  workmen  in  the  building  trades  on  outside  work  in 
the  colder  weather,  teamsters  and  car  drivers  who  are  constantly 
exposed  to  the  weather,  even  when  the  work  is  not  excessive,  may  con- 
sume a  diet  yielding  4,000  or  even  5,000  Cal. 

Special  Diets.  With  such  facts  as  the  above  in  mind  it  is  not  diffi- 
cult to  understand  why  nutrition  with  a  single  article  of  food  is  unsat- 
isfactory. Assume,  for  example,  the  case  of  a  diet  of  potatoes  of 
which  the  edible  portion  shows  in  the  mean  about  these  per  cent  values : 
protein  2.2,  fat  0.1,  carbohydrates  18.4.  One  hundred  grams  of  pota- 
toes would  yield  then  the  following : 

Protein    2.2  gm.  9.0  Cal. 

Fat    0.1  0.9 

Carbohydrate     18.4  75.4 

A  pound  of  potatoes  would  furnish,  therefore,  386  Cal.,  and  6  to  8 
pounds  would  have  to  be  consumed  to  furnish  energy  for  ordinary 
work.  The  protein  in  this  would  be  considerably  below  the  amount 
which  has  generally  been  held  necessary,  while  the  fat  is  scarcely 
appreciable.  The  storage  of  energy  on  such  a  diet  would  be  practically 
impossible. 

On  the  other  hand,  a  diet  of  lean  meat,  round  steak,  for  example, 
would  be  almost  as  bad.  This  averages  about  20.9  per  cent  of  protein 
and  10.6  per  cent  of  fat,  from  which  100  grams  would  furnish: 

Protein    20.9  gm.  85.7  Cal. 

Fat    10.6  98.6 

1843 

A  pound  would  furnish  835  Cal.  and  about  3  to  4  pounds  would  have 
to  be  consumed  for  support  of  the  body  daily.  While  the  fat  in  this 
would  be  proper  the  protein  would  amount  to  275  grams  at  least,  and 
make  the  work  of  excretion  extremely  difficult. 

As  a  third  case  a  diet  of  the  small  white  beans  may  be  considered. 
We  have  here  protein  22.5,  fat  1.8,  carbohydrate  59.6.  In  100  grams, 
therefore, 

Protein    22.5  gm.  92.2  Cal. 

Fat    1.8  16.7 

Carbohydrate    59.6  244.3 

353-2 


37^  PHYSIOLOGICAL    CHEMISTRY. 

A  pound  would  furnish  about  1600  Cal.  and  2  pounds  would  cover  the 
needs  of  the  body  practically.  The  proteins  in  this  would  be  but 
slightly  excessive,  while  the  same  would  be  true  of  carbohydrates. 
The  trouble  is  with  the  deficiency  in  fat.  Notice  how  easily  this  may 
be  corrected.  A  pound  and  a  half  of  beans  cooked  with  one  fourth 
pound  of  fat  pork  will  yield  over  3,000  Cal.  and  furnish  a  diet  easily 
assimilated  by  men  at  moderate  work.  What  is  said  of  the  bean  is 
practically  true  of  the  pea ;  each  one  approaches  in  value  a  mixed  meat 
and  cereal  diet. 

Required  Protein.  A  much  debated  question  is  that  of  the  actual 
protein  requirement,  supposing  the  other  food  elements  sufficiently 
abundant.  The  standards  given  above  have  not  gone  unchallenged. 
Several  observers  have  described  metabolism  tests  in  which  less  than 
one  half  the  120  or  more  grams  of  protein,  usually  considered  neces- 
sary in  the  daily  food,  appears  to  be  sufficient  for  all  needs  and  able 
to  maintain  the  body  in  nitrogen  equilibrium.  Most  of  these  experi- 
ments have  been,  however,  too  short  to  really  prove  much  definitely. 

But  recently  very  elaborate  and  long  continued  investigations  on 
groups  of  men  have  been  described  by  Chittenden  in  which  the  evi- 
dence in  favor  of  a  low  protein  requirement  is  put  in  an  entirely  new 
light,  and  in  which  it  is  also  shown  that  the  3,000  large  calories  of 
energy  in  our  food  is  more  than  necessary  for  ordinary  practical  needs. 
In  a  group  of  five  professional  men  Chittenden  found  an  average  nitro- 
gen liberation  corresponding  to  the  metabolism  of  something  over  46 
gm.  of  protein  daily  and  an  average  energy  value  in  the  whole  food  of 
about  2,300  calories  through  a  period  of  six  to  nine  months.  In  a 
group  of  thirteen  soldiers,  taking  abundant  exercise,  there  was  a  daily 
consumption  of  food  having  an  average  value  of  about  2,600  calories, 
and  an  average  protein  consumption  of  about  56  gm.  through  five 
months,  fall,  winter  and  early  spring.  In  a  group  of  seven  student 
athletes  a  protein  consumption  of  about  61.5  gm.  daily  with  a  total 
food  consumption  equivalent  to  about  2,575  calories  was  observed. 

In  all  these  cases  the  tests  were  continued  long  enough  to  bring  the 
men  into  practical  nitrogen  equilibrium  with  good  physical  condition 
and  good  general  health.  For  this  reason  they  deserve  the  fullest 
attention  and  study.  It  is  the  opinion  of  the  author  of  the  experi- 
ments that  increased  food  consumption,  so  far  from  being  necessary, 
is  even  in  most  cases  a  detriment,  since  it  calls  for  a  large  amount  of 
extra  internal  work,  in  the  liver  and  kidneys  especially,  in  metabolizing 
the  digestion  products  and  in  removing  the  waste.  This  is  certainly 
a  consideration  of  some  moment.     How  far  these  findings  may  be 


THE    ENERGY    EQUATION.  379 

applied  to  the  case  of  men  at  hard  work,  in  the  open,  in  cold  weather, 
remains  to  be  tried.  Chittenden's  results  are  especially  interesting 
with  respect  to  the  necessary  nitrogen;  but  for  the  hard-working 
man  probably  more  fat  and  more  carbohydrate  will  always  be  found 
desirable. 


INDEX 


Abnormal  colors  in  urine,  325 
Absorption  analysis,  194 

cells  for  spectroscope,  200 

from  stomach,  143 

ratios,  201 
Acetic  acid,  40 

fermentation,   115 
Acetoacetic  acid  in  urine,  322 
Acetone  in  urine,  322 
Achroodextrin,  27 
Acid  albumin,  yy 

fermentation,  159 
of  urine,  349 
Acid,  acetic,  40 

alloxyproteic,  302 

amino-acetic,  61 
caproic,  62 
glutaric,  62 
isobutylacetic,  62 
propionic,  62 
succinic,  62 
valeric,  62 

antoxyproteic,  302 

arabic,  38 

arabonic,  18 

arachidic,  40 

aspartic,  62 

behenic,  40 

butyric,  40,  119 

capric,  40 

caproic,  40 

caprylic,  40 

carbonic,  64 

cholalic,  265 

cholanic,  265 

choleic,  265 

chondroitin  sulphuric,  89 

cresyl  sulphuric,  303 

dextronic,  18 

elaidic,  46 

erythritic,   18 

fellic,  265 

formic,  40 

glutaminic,   62 

glyceric,  18 

glycerophosphoric,    48 


Acid,  glycocholic,  264 

glycollic,    18 

hippuric,  301,  334 

hypogseic,  41 

indoxyl  sulphuric,  303 

lactic,  117 

in  stomach,  134 

lauric,  40 

linoleic,  41 

lithofellic,  265 

mannonic,  18 

margaric,  40 

myristic,  40 

nucleic,  86 

cenanthylic,  40 

oleic,  41 

oxalic,  18 

oxyphenyl  amino  propionic,  63 

oxyproteic,  301 

palmitic,  40 

parabanic,  308 

paralactic,  281 

pelargonic,  40 

pentoic,  40 

phenyl  amino  propionic,  63 

phenyl  sulphuric,  303 

phosphoric,  urinary,  304 

picric,  56 

propionic,  40 

pyrrolidine  carboxylic,  62 

ricinoleic,   41 

saccharic,  18 

sarcolactic,  119 

skatoxyl  sulphuric,  303 

stearic,  40 

tartaric,  18 

tartronic,  18 

taurocholic,  264 

thiolactic,  281 

trioxyglutaric,   18 

undecylic,  40 

uric,  297 

valeric,  40 
Acidity  of  gastric  juice,  137 
Acids  in  fats,  40 
Acid  zone,  158 


380 


INDEX. 


381 


Acrose,  19 
Activators,  ioi,  157 
Addiment,  225 
Adenine,  87 
Adipocere,  45 
Adrenalin,  273 
Agar-agar,  38 
Agarose,  27 
Agave  sugar,  27 
Agglutinins,  221 
Air,  12 

tests,  13 
Alanine,  62 

Alanylglycylglycine,  149 
Albumin,  51 
Albuminates,  77 
Albuminoids,  53,  90 
Albumin  in  urine,  314 
Albumins  proper,  66 
Albumoses,  80,  81 
Alcoholic  fermentation,  112,  113 
Alcohol  in  wine,  113 
production,  113 
test  for,  113 
Aldopentoses,  18 
Alexins,  224 
Alkali  albumin,  77 
Alkalies  and  protein,  61 
Alkaline  zone,  158 
Alkaloid  reagents  and  proteins,  56 
Alloxyproteic  acid,  302 
Alpha  naphthol  test,  23,  58 
Amboceptors,  226 
Amino  acids,  61 

as  digestive  products,  150 
Aminocaproic  acid,  62 
propionic  acid,  62 
valeric  acid,  62 
Ammoniacal  copper  solution,  29 
Ammonia,  determination  of,  330 
in  urine,  296 
in   water,   10 
Ammonium  cyanate,  296 
Amniotic  fluid,  233 
Amorphous  phosphates  in  urine,  353 
Amount  of  acid  in  stomach,  135 

sugar  in  urine,  320 
Amphopeptone,  83 
Amygdalin,  106 
Amylodextrin,  37 
Amyloid  degeneration,  92 

substance,   92 
Amylopsin,  103 


Amylase,  103 
Amylose,  33 
Analysis,  spectrum,  197 
Analyses,  ash  of  milk,  240,  241 
ash  of  muscle,  283 
bile,  263 
blood,  176 
bone  ash,  286 
cells  of  thymus,  233 
cerebrospinal  liquid,  277 
colostrum,  241 
feces,  164,  166 
gall  stones,  270 
hydrocele  fluid,  233 
lymph,  230 
meat  extract,  284 
milk  of  cow,  236 
mother's  milk,  245 
muscle,  278 

peritoneal  transudate,  233 
pleural  transudate,  233 
pus  cells,  234 

serum,  233 
spermatic  fluid,  275 
urine,  292 
Animal  foods,  293 

internal  work  of,  373 
starch,   36 
Animals  and  plants,  3 
Anti  bodies,  chemical  nature,  223 

development  of,  220 
Anti  body  defined,  217 
Anti  group,  81 
Antipeptone,  83 
Antitoxins,  218 
Antoxyproteic  acid,  302 
Apparatus  for  freezing  point,  207 
Arabinose,  18,  20,  38 
Arabitol,  18 
Arabonic  acid,  18 
Arachidic  acid,  40 
Arginine,  61,  84 
Argon  in  blood,  190 
Aromatic      products      from      intestinal 

putrefaction,  160 
Artificial  purification  of  water,  9 
Ash  in  tissues,  14 

of  milk,  240 
Aspartic  acid,  62 
Assay  of  pepsin,  131 
Atwater,  food  standards,  376 
Autodigestion,  256 
Autolysis,  bacterial   products   from,  257 


382 


INDEX. 


Autolysis,  importance  of,  257 

organic  acids-  from,  257 

pancreas,  272 

protein  in,  257 
Autolytic  fermentation,  255 

Bacteria   in   feces,    163 

in  urine,  348 

lactic  acid,  117 
Bacterial  process,  158 

purification  of  water,  9 
Bactericidal  products  of  autolyses,  257 
Bacteriolysins,  219 
Bacteriolytic  processes,  117 
Bases  in  body,  16 
Beckmann  apparatus,  207 
Beef  extract,  280 

composition,  93 

pancreas,  extracts  from,  146 
Beeswax,  49 
Beet  sugar,  25 
Behavior  of  trypsin,   145 
Behenic  acid,  40 
Benedict  and  Gephart,  urea  method,  330 

total  sulphur  in  urine,  339 
Benjamin  Thompson,  6 
Benzoates  and  hippuric  acid,  301 
Benzoic  acid,  106 
Bernard,  C.  L.,  5 
Berzelius,  4 

Bicycle  rider,  food  and  work,  376 
Bile,  262 

acids,  265 

in  feces,  169 
optical  rotation,  266 

colors  in  feces,  174 

composition,  263 

concretions,    270 

emulsification  by,  269 

pigments,  266 
Bilicyanin,  271 
Bilifuscin,  271 
Bilihumin,    271 
Biliprasin,  271 
Bilirubin,  188,  263,  267 
Biliverdin,   188,  265,  267 
Biology,  field  of,  2 
Bismuth  test,  23 
Bitter  almonds,  106 
Biuret,  57 

reaction,  57 
Blood, 175 

albumin,  66 


Blood,  analyses,  176 

and  bile  pigments,  185 

anti  bodies  in,  218 

ash  of,  14 

casts,  346 

cholesterol  in,   191 

conductivity,  212 

corpuscles  in  urine,  342 

cryoscopy,   206 

freezing  point,  206 

gases  of,   190 

in  tissue,  191 

in  urine,  326 

lecithins  in,  191 

optical  properties,  193 

osmotic  pressure,  204 

phagocytes  in,  216 

salts  of,  190 

self  preservation,  216 

serum  tests,  219 

sugar  in,  189 

tests,  clinical,  201 

transfusion,  192 
Boas'  reagent,  134 
Body,  bases  in,  16 

composition  of,  7 
Bogg's  coagulometer,  180 
Bone,  285 

ash  of,  14,  286 

gelatin  from,  90 

glue  from,  90 

marrow,  287 

ossein  in,  256 
Brain  and  nerve  substance,  276 
Bran,  pentose  in,  20 
Bread,  composition,  94 

fermentation  in,  118 
British  gum,  35 

Brunner's  glands,  juice  from,  156 
Buchner,  ferments,  99 
Bunge,  248 

Burchard-Liebermann  test,  50 
Butter,  46 

composition,  238 
Butter  milk,  242 
Butyric  acid,  40,  119 

fermentation,  117 

Cadaver  wax,  45 

Calcium  and  magnesium  salts  in  urine, 
293 

in  body,  8 

oxalate  in  sediment,  352  ' 


INDEX. 


333 


Calcium  sulphate  in  sediment,  352 

Calculation  of  food  energy,  368,  369 

Calculi,  354 

Calves  stomach,  rennet  in,  108 

Calorie,  definition,  366 

Calories  in  foods,  93,  94 

Cane  sugar,  25 

group,  25 
Caproic  acid,  40 
Caprylic  acid,  40 
Carbohemoglobin,   186 
Carbohydrates,  17 

changes  in  liver,  253 

digestion,  103,  152 

group  in  proteins,  81 

heats  of  combustion,  367 

in  urine,  304 
Carbonates  of  body,  15 
Carbon  balance,  357 

dioxide  and  plant-life,  2 

in  body,  8 

monoxide  hemoglobin,  184 
Carnine,  280 
Carnitine,  280 
Carnosine,  280 
Cartilage,  287 

ash  of,  14 

gelatin  from,  90 

mucoid  in,  89 
Casein,  72 

in   feces,   173 

preparation,  239 
Caseoses,  81 
Casts  in  urine,  345 
Catalytic  action,  98 
Cat  fat  crystals,  43 
Cell  globulin,  69 
Cells,  conductivity,  214 

epithelium  in  urine,  343 

in  general,  249 

lymph,  233 
Celluloid,  30 
Cellulase,  104 
Cellulose,  38 

in  feces,  171 
Centrifuge,  uses  of,  341 
Cereals,  composition,  94 
rospinal  liquid,  276 
Character  of  antibodies,  218 
Charts,  Tallquist's,  203 
Cheese,  composition,  93 
Chemical  nature  of  anti  bodies,  223 
Chemistry  of  milk,  238 


Chittenden,  53 

preparation  of  pepsin,  130 

protein  classification,  81 

protein  requirement,  378 
Chlorides,  determination  of,  2>2>7 

in  water,   10 

of  body,  8,  15 

tests  for,  10 
Chlorophyll-bearing   plants,    2 
Cholagogues,  268 
Cholanic  acid,  265 
Choleic  acid,  265 
Cholesterol,  49,  270 

in  blood,  191 

in  feces,  169 

in  protoplasm,  250 

optical    rotation,    271 
Choline,  48 
Cholalic  acid,  265 
Chondroitin,  89 

sulphuric  acid,  89,  287 
Chondromucoid,  89,  287 
Chromophoric  group,  136 
Chyle,  232 

Classification  of  proteins,  52 
Clinical  blood  tests,  201 

uses  of  hematocrit,  211 
Clumping,  bacteria,  222 
Clupein,  75 

Coagulated  albumins,  76 
Coagulating  proteins,  69 
Coagulation  of  blood,  177 

tests,  54 
Coagulometer,  180 
Coefficient,  isotonic,  208 
Co-enzymes,   101 
Cohnheim,  156 

theory  of  protein  metabolism,  290 
Collagen,  90 

from  muscle,  279 
Collodion,  39 

Coloring  matter  in  urine,  323 
Color  of  urine,  292 
Colostrum,  241 
Combustion,  heats  of,  367 
Commercial  bile  salts,  269 

pepsin,  109,  130 
Complement,  225 
Complementoids,  228 
Component  groups  in  proteins,  60 
Composition  of  body,  7 

of  cells,  249 

of  lymph,  230 


3§4 


INDEX. 


Composition  of  milk,  237 
Conductivity  cells,  214 
of  blood,  212 
of  urine,  309 
Congo  red  test,  133 
Coniferin,  107 
Conjugated  proteins,  54 
Conservation  of  energy,  6 
Conversion  of  starch,  37 
Corpuscles,  number  in  blood,  211 
Count  Rumford,  6 
Cow's  milk,  236 
Creatine,  279 
Creatinine,  279 

determination,  333 
from  urine,  300 

reducing  power,  307 
Crude  fat  in  feces,  168 
Cryoscopy,  206,  312 
Crystallin,  69 
Crystals  of  fats,  43 

of  hemin,  181 
Curtius,  64 
Cystin,  92,  262 

calculi,  355 

in  urine,  351 
Cytase,  104 
Cytosine,  87 
Cytotoxins,  219 
Dare's  hemoglobinometer,  202 
Dehydrocholeic  acid,  265 
Denatured  proteins,  77 
Derived  products,  proteins,  53 

protein,  54 
Despretz,  5 

Destruction  of  glycogen,  255 
Detection  of  free  acid  in  stomach,  133 
Determination  of  acid  in  stomach,  135 

of  albumin  in  urine,  314 

of  ammonia  in  urine,  330 

of  blood  colors,  197 

of  chlorides  in  urine,  357 

of  creatinine  in  urine,  333 

of  digestive  power,  pepsin,  131 

of   electrical  conductivity,  212 

of  fats,  47 

of  fats  in  milk,  244 

of  feces  fat,  169 

of  freezing  point,  207 

of  hemoglobin,  203 

of  hippuric  acid,  334 

of  nitrogen  in  feces,  172 

of  nitrogen  in  urine,  327 


Determination  of  osmotic  pressure,  209 
of  pepsin,  139 

of  phosphates  in  urine,  335 
of  proteins,  59 

of  milk,  244 
of  purine  in  urine,  333 
of  specific  gravity,  313 
of  sugar,  27 
in  milk,  244 
in  urine,  320 
of  sulphates  in  urine,  339 
of  total  sulphur  in  urine,  339 
of  urea  in  urine,  328 
of  uric  acid  in  urine,  332 
Deutero  albumose,  83 
Dextrin,  35 

from  starch,  123 
Dextronic  acid,  18 
Diabetes  mellitus,  305 
Diagram  of  spectroscope,  194    ' 

Wheatstone  bridge,  212 
Dialyzer,  67 
Diastase,  97,  103 

action  of,  123 
Diazo  reaction,  162,  326 
Diet  and  feces,  165 
Dietaries,  376 
Diets,   special,  377 
Digestion,  96 

of  fat's  in  stomach,  143 
of  starch,  122 
pancreatic,  144 
peptic,  109,  127 
salivary,  121 
tryptic,  no 
Digestive  extracts,  146 
Diglycylglycine,   149 
Dilution  test,  315 

Dimethylaminoazobenzene  test,  133 
Diose,  18 
Dioxyacetone,   18 
Direct  vision  spectroscope,   194 
Disaccharides,  17 
Distearin,  41 

Distribution  of  food  energy,  368 
of  heat  energy,  370 
of  nitrogen  in  urine,  295 
Donne's  test,  343 
Dulong,  5 

Edestan,  69 
Edestin,  69 
Edible  fats,  41 


INDEX. 


3»! 


Effect  of  work,  371 
Egg  albumen,  67 

and  pepsin,   131 
Ehrlich  reaction,  162 
Ehrlich's  theory,  224 
Elaidic  acid,  47 
Elaidin,  47 
Elastin,  92 
Electrical  conductivity  of  blood,  212 

of  urine,  309,  310,  311 
Elements  in  body,  7,  8 
Emulsin,   106 
Emulsions,  42,  269 
Endothermal  reactions,  2 
End  products  of  digestion,  150 

of  metabolism,  289 
Energy  balance,  289 
equation,  366 
of  food,  distribution,  368 
Enterokinase,  157 
Enzymes,  96 

as  catalytic  agents,  100 

of  stomach,  127 
Epinephrin,  273 
Epithelium  in  urine,  343 
Erepsin,  no,  156 
Erg,  definition,  367 
Erythritic  acid,   18 
Erythrodextrin,  yj 
Erythrogranulose,  37 
Erythrol,  18 
Erythrose,  18 
Esbach  albuminometer,  139,  315 

reagent,  139 
Ethereal  sulphates,  162,  261 
Ewald  test  meal,  132 
Examination  of  stomach  contents,  132 
Excretion  by  skin,  363 

gaseous,  356 

of  alkali  salts,  293 

of  calcium  and  magnesium,  293 

of  nitrogen,  289 

of  phosphorus,  304 

of  sulphur,  302 
External  work  equivalent,  372 
Extinction  coefficient,   igg 
Extract  of  meat,  283 
Extracts  from  yeast,  114 
Extractives  from  muscle,  277 
Exudations,  232 

Fat   and  chyle,  232 
crystals,  43 
26 


Fat  from  muscle,  279 

globules,  239 

in  urine,  351 

in  feces,  164 

in  foods,  93,  94 

of  milk,  238 
Fats,  40 

from  proteins,  44 
sugars,  44 

heats  of  combustion,  367 

in  blood,  191 

in  body,  44 

in  pancreatic  digestion,   154 

solubility,  44 

splitting  of,  107 
Fatty  acids,  40 
Faulhorn  experiment,  375 
Feces,  161,  163 

amount  of,  164 

bile  acids  in,  169 

blood  and  pus  in,  174 

carbohydrates  in,   170 

cellulose  in,  171 

composition,  164 

from  various  foods,  165 

lecithin  in,  168,  170 

nitrogen  in,  172 

proteins  in,  173 

starch  in,  170 

sugar  in,  171 
Fehling  reduction,   28 

test,  22 

urine,  318 
Fellic  acid,  265 
Fermentation,  acetic,  115 

alcoholic,  113 

autolytic,  255 

butyric,  97,  117 

in  intestines,  158 

lactic,  97,    117 

mucous,  119 
Ferments,  96 

classification,  102 
Ferric  chloride  test,  323 
Fibrin,  70,  177 

digestion  of,  147 
Fibrinogen,  70 
Fibronoses,   81 

Fick  and  Wislicenus  experiment,  375 
Fields  of  study,  2 
Filter  paper,  38 
Fischer,  64,  225 

nomenclature  of  purines,  298 


386 


INDEX. 


Fish,  composition,  93 
Fission  fungi,  112 
Fleischl  hemometer,  201 
Flesh  bases,  279 
Flour,  composition,  94 
Fluorine  in  body,  8 
Folin,  ammonia  in  urine,  331 

creatinine  method,  334 

sulphate  method,  339 

theory  of  protein  metabolism,  290 

urea  method,  330 

uric   acid  method,  332 
Food  and  work,  6 

consumption  and  muscular  work,  375 

of  plants,  3 
Foods,  relation  to  feces,  165 
Food  stuffs,  93 

Formaldehyde  condensation,   2 
Formic  acid,  40 
Formulas   for  hemoglobin,   187 

spectrophotometry,  199 
Fraunhofer  lines,  193 
Free  acid  in  stomach,  132 
Freezing  point  of  blood,  206 

urine,  312 
Fructose,  18,  24 
Fruit  sugar,  20 

Fuel  value  of  foods,  93,  94,  367 
Functions  of  bile,  268 

liver  cells,  251 

lymph,  231 
Fungi  and  fermentation,  112 

in  urine,  347 
Furfuraldehyde,  20 
Furoaniline,  20 

Gadus-histone,   75 
Galactose,  24,  105,  240 
Gallstones,  49,  270 
Gaseous  excretions,  356 
Gases  in  air,   12 

of  blood,  190 
Gastric  juice,  126 
acidity,  137 
titration  of,  138 
Gelatin,  90,  285 

tests  for,  91 

uses,  91 
General  composition  of  urine,  291 

relations,  1 
Gland,  thyroid,  274 
Gliadin,  73 
Globin,  74 


Globulinoses,  81 
Globulins,  68 

in  urine,  315 
Glucase,  104 
Gluco-proteids,  88 
Glucosamine,  63 
Glucose,  21 

from  starch,  21 

in  blood,  189 

reducing  power,  28 
Glucoses,  18 
Glucosides,  20 
Glucoside  reactions,  106 
Glucoronic  acid  in  urine,  322 
Glue,  90 

Glutaminic  acid,  62,  84 
Glutelins,  74 
Gluten,  73,  94 
Glutenin,  73 
Glutin,  go 
Glyceric  acid,  18 
Glyceraldehyde,    18 
Glycerol,  18,  47 
Glycero-phosphoric  acid,  48 
Glycerose,  18 
Glyceryl  butyrate,  46 

caproate,  46 

oleate,  46 
Glycine,  61 
Glycocoll,  61,  264 
Glycogen,  36,  280 

destruction,  255 

formation,  253 

in  flesh,  281 

in  protoplasm,  250 

stored  in  liver,  254 
Glycol,  18 
Glycollic  acid,  18 
Glycocholic  acid,  264 
Gmelin,  4 
Gmelin's  test,  267 
Goitre  and  iodine  compounds,  274 
Gower's   hemoglobinometer,  203 
Graham  dialyzer,  67  > 

Grain  composition,  94 
Granular  casts,  346 
Granulose,  33 
Grape  sugar,  21 
Group,  immune,  227 

zymotoxic,  228 
Groups  in  protein,  60 
Guaiacum  test,  180,  327 
Guanine,  87 


INDEX. 


387 


Guenzberg's  reagent,  133 
Gum  arabic,  38 

British,  35 
Gums,  37 
Gun  cotton,  39 

Hair,  keratin  from,  288 
Hamburger,  209 
Hammarsten's  test,  268 
Haptophorous  group,  226 
Hard  water,  8 
Heart,  ash  of,  14 
Heat  and  food  stuffs,  6 

energy,  distribution,  370 

mechanical    equivalent,   367 

of  friction,  6 

production   incidental,  374 

radiation,  371 

unit,  definition,  366 
Heats  of  combustion,  367 
Hematin,  187 

spectrum,  196 
Hematocrit,  clinical  uses,  211 

methods,  210 
Hematogen,  73 
Hematoidin,  188 
Hematolin,  187,  188 
Hematoporphyrin,  187 
Hematuria,  326 
Hemi  group,  81 
Hemin  crystals,   181 
Hemochromogen,   187,    188 
Hemoglobin,  74,  181 

analysis,  182 

combinations,    182 

crystals,  183 

specific  rotation,  182 
Hemoglobins,  87 
Hemoglobinometer,  Dare's,  202 

Gower's,  203 
Hemoglobinuria,  326 
Hemometer,  Fleischl's,  201 
Hemolysins,  219 
Heteroalbumose,  82 
Hexitols,  18 
Hexone  bases,  61 

in  digestion  products,   149 
Hexoses,  18,  20 
Hippuric  acid,  301 

determination,  334 
in  sediment,  352 
Hirn,     comparison     between     man     and 
machine,  371 


Histidine,  61 

Histones,   74 

Historical  sketch,  4 

History  of  fermentation,  97 

Hofmeister,  64 

Hog  pancreas,  extracts   from,  146 

Hoppe-Seyler,  5 

Horn,   composition,  93 

keratin   from,  288 

substance,  92 
Human  fat,  47 

milk,  245 
Hyaline  casts,  346 
Hydrazones,  20 
Hydrocele  fluid,  233 
Hydrochloric  acid  in  stomach,  126 
Hydrocyanic  acid,  106 
Hydrogen  in   body,   8 

peroxide  test,  181 
Hydrolysis    of   proteins,   60 

starch,  22,  122 
Hydrolytic  reactions,  102 
Hypogaeic  acid,  41 
Hypoxanthine,    87 

Ichthulin,  73 
Immune  body,  226 

group,  227 
Immunization,  219 
Important  early  works,  5 

fats,  45 
Indestructibility  of  matter,  6 
Index,  opsonic,  222 
Indicators  and  stomach  contents,  136 

theory  of,  136 
Indican,  151,  161,  324 
Indol,   147,   151,  160 
Indoxyl,   151,  161 
Inorganic  elements,  7 
Inosite,  281 
Insoluble  ferments,  99 
Intermediary  body,  226 
Internal  work  of  animal,  373 
Intestinal  bacteria,  159 

changes,  158 

juice,  155 
Inulase,   104 
Tnulin,  24,  35 
Invertasc,  25,   105 
Invertin,   105 
Invert  sugar,  25 

reducing  power,  28 
Investigations,  early,  5 


388 


INDEX. 


Iodine   in  body,  8 

in  thyroid,  274 

test,  34 
Iodothyreoglobulin,  274 
Iodothyrin,  275 
Iron  in  bile,  267 

in  body,  8 

masked,  86 
Isinglass,  91 
Isocholesterol,  49 
Isodynamic  ratios,  374 
Isolation  of  pepsin,  129 
Isomalt'ose,  27 
Isotonic  coefficient,  208 

Joule,  6 

Juice,  intestinal,  155 
pancreatic,  144 

Kelling's  test,   135 
Kephir,   26,    118 
Keratin,   92,   288 
Ketopentose,  18 
Kidney,  ash  of,  14 
Kinases,  101,  157 
Kinds  of  ferments,  102 
Kinetic  energy  of  food,  368 
Kjeldahl  test,  327 
Koeppe's  hematocrit,  210 
Koprosterin,  271 
Kruess  spectrophotometer,  i( 
Kuehne,  5 

protein  classification,  81 
Kumyss,  118 
Kyrine,  84 

Laborers,  dietaries,  376 
Laccase,  116 
Lactalbumin,  67,  239 
Lactic  acid,  281 

bacteria,  117 

tests  for,  135 
fermentation,  117 
Lactase,  105 

Lactose,  25,  26,  105,  240 
Landwehr's  animal  gum,  88 
Lanolin,  49 
Laplace,  5 
Lard,  46 

Laurent  polariscope,  30 
Laurie  acid,  40 
Lavoisier,  4 
Lead  hydroxide  test,  58 


Lecithan,  48 
Lecithin,  48,  68 
Lecithins  in  blood,  191 

in  cells,  250 

in  feces,  168 
Legumin,  74 
Lehmann,  5 
Leucine,  62,  92 

as  urine  sediment,  350 

tests  for,  147 
Leucocytes,  231,  233 
Leucylproline,  149 
Leuwenhock,  96 
Levulose,  24 
Lieberkuehn's  glands,  juice  from,  155 

jelly,  78 
Liebig,  4 

theory  of  fermentation,  98 
Lignocellulose,  39 
Linoleic  acid,  41 
Lipase,  107,  126,  154 
Lithofellic  acid,  265 
Liver  and  poisons,  258 

ash  of,  14 

autolysis,  256 

chemical  changes,  252 

chemistry  of,  249 

ethereal  sulphates,  261 

fats  in,  251 

formation  of  urea,  259 
uric  acid,  260 

glycogen  in,  251 

iron  in,  252 

lecithin  in,  251 

mineral  substances,  252 

protein  in,  251 

synthetic  processes,  259 

work  of  cells,  252 
Loewe  solution,  29 
Loss  of  free  acid  in  digestion,  142 
Lymph,  230 

amount,  231 

composition,  230 

functions,  231 
Lymphagogues,  231 
Lysine,  60,  84 

Magnesium  in  body,  8 

phosphate  in  urine,  353 
Malondiamide,  57 
Malt,  103 
Maltase,   104 
Malt  extract,  123 


INDEX. 


;89 


Maltodextrin,  37 
Maltose,  25,  104 

reducing  power,  28 
Malt  sugar,  26,  123 
Margarin,  45 

Manufacture  of  starch,  33 
Maple  sugar,  25 
Market  milk,  236 
Marrow,  287 
Masked  iron,  86 
Mayer,  6 

Margaric  acid,  40 
Meal,  composition,  94 
Meat,  composition,  93 

extract,  283 
Mechanical  equivalent  of  heat,  367 
Melibiose,  27 
Melitose,  27 

Meyer,  blood  gases,  190 
Metabolism  experiments,  360-362 

theories  of,  290 
Methemoglobin,    186 

spectrum,  197 
Metaproteins,  80 
Methyl  orange  indicator,  136 

violet  test,  133 
Microorganisms   in   fermentation,  9J 
Milk,  236 

albumin,  67 

ash  of,   14 

composition,  95 

curdling  ferment,  143 
of,  108 

fat  in,  238 

flavors,  247 

human,  245 

modified,  246 

mother's,  245 

of  ass,  248 

of  bitch,  248 

of  elephant,  248 

of  goat,  248 

of  mare,  248 

of  sow,  248 

origin  of,  237 

preservatives,  244 

salts  of,  240 

sugar,  24,  26,  240 

reducing  power,  28 
Milton's  reagent,  56 

test  in  digestion,  14X 
Mineral   matter-,    in    blood,    177 

residues  of  organs,  14 


Mineral   substances  in  milk,  240 
Modified  albumin,  76 

milk,  246 
Molds  in  urine,  348 
Molisch  reaction,  83 

test,  23,  58 
Mannitol,  18 
Mannonic  acid,  18 
Monosaccharides,  17 
Monoses,  17 
Monostearin,  41 
Moore's  test,  317 
Mother  of  vinegar,  115 
Mucin  bands  in  urine,  344 

in  saliva,  121 

in  urine,  316 
Mucins,  88 
Mucoid  bodies,  89 
Mucors,  112 

Mucous  fermentation,  119 
Mucus  in  urine,  342 
Murexid  test,  331 
Muscle,  ash  of,  14 

extraction,  71 

plasma,  71 

sugar,  281 

substance,  277 
Musculin,  278 
Mutton  tallow  crystals,  43 
Mycose,  27 
Myogen,  70,  278 
Myosin,  70,  107,  278 
Myosinogen,  70 
Myosinoses,  81 
Myricin,  49 
Myristic  acid,  40 

Nails,  keratin  from,  288 
Natural   fats,  40 

purification  of  water,  9 

waters,  8 
Native  albumins,  53,  65 
Nature  of  bile,  268 
Neutral   sulphur,  262,  303 
Nicol  prism,  31 
Nitrates  in  water,   11 
Nitric  oxide  hemoglobin,   185 
Nitrites  in  water,  11 
Nitrocellulose,  39 
Nitrogen  balance,  357 

excretion   of,  294 

-free  extractives  from  muscle,  280 

in   blood,    190 


39° 


INDEX. 


Nitrogen  in  body,  8 

in  feces,  164,  172 

of  urine,  distribution  of,  295 
Normal  colors  in  urine,  333 

feces,  163 

reduction,   307 
Nucleates,   86 
Nucleic  acid,  85,  86,  298 
Nuclein,  85,  250 
Nucleo-albumin,  71 
Nucleo-histone,  75,  86 
Nucleo-proteids,  85 
Nucleus,  249 

Number  of  corpuscles,  211 
Nutrients,  7 
Nutrose,  72 
Nuts,  composition,  94 

Occurrence  of  metals  in  body,  8 

Odor  of  urine,  292 

Oil  of  bitter  almonds,  106 

Oleic  acid,  41 

Olein,  45 

Oleomargarin,  46 

composition,  93 
Opsonins,  222 
Opsonic  index,  222 

treatment,  223 
Optical  properties  of  blood,  193 

rotation,  30 

sugar  tests,  30 
Organic  acids  by  bacteria,   141 
from  liver,  256 
in  stomach,  134 

chemistry  and  agriculture,  4 
pathology,  4 
physiology,  4 

matter  of  bones,  286 
Organized  ferments,  99 

sediments,  341 
Organs  of  body,  ash  of,  14 
Origin  of  fats,  44 
Osazones,  21 
Osborne,  53,  69 
Osmotic  pressure,  204 
cell,  205 

tension,  209 
Osones,  21 
Ossein,  90,  286 
Outline  of  topics,  6 
Oxalate   calculi,   355 
Oxalic  acid,  18 
Oxaluramide,  57 


Oxamide,  57 
Oxidase  enzymes,  115 
Oxidases,    115 
Oxidation  reactions,   111 

test's,  10 

time  and  place  of,  364 

value  of  copper  solutions,  28 
Oxybutyric  acid  in  urine,  322 
Oxygen  absorption  by  blood,  184 

in  blood,  190 

in  body,  8 

liberation  of  by  plants,  2 
Oxyhemoglobin,  183 

spectrum,    195 
Oxyproteic  acid,  295,  301 
Oysters,  composition,  93 
Ozone  in  air,  13 

Palmitic  acid,  40 
Palmitin,  45 
Pancreas,  272 

ash  of,  14 

autolysis,  272 
Pancreatic  diastases,  153 

digestion,  144 
of  fats,  154 

starch,  153 

ferments,  no 

juice,   144 
Pancreatin  and  milk,  243 
Paracasein,  72 
Paralactic  acid,  281 
Parasitic  plants  like   animals,  3 
Parathyroids,  importance  of,  275 
Pasteur,  5,  97 

theory  of  fermentation,  98 
Pavy  method,  321 

solution,  29 
Pawlow,  126 
Payen,  97 

Peas,  composition,  94 
Pectase,  104 
Pectin,  104 
Pectinase,  104 
Pelargonic  acid,  40 
Pentitols,  18 
Pentoic  acid,  40 
Pentosans,  20 
Pentoses,  18,  19 
Pepsin,  108,  126 

amount  of,  139 

peptone,  150 

preparation,  129 


INDEX. 


391 


Pepsinogen,    108 
Peptic  digestion,  127 

products  of,  140 
Peptones,  80,  81,  83,  108,  141 

in   urines,  316 
Percentage  variations  in  urine,  291 
Peritoneal  transudates,  233 
Permanent   hardness,   9 
Permanganate  test,   11 
Peroxidases,  116 
Persoz,  97 

Pfeiffer  phenomenon,  224 
Pflueger  theory  of  protein  metabolism, 

290 
Phagocytes,  216 
Phenylalanine,  63 
Phenylalanylglycylglycine,  149 
Phenyl  glucosazone,  23 
Phenyl  hydrazine  test,  21,  23 

in  urine,  319 
Phenol  in  intestinal  changes,  160 
Phenol-phthalein   indicator,   136 
Phenomenon,  Pfeiffer's,  224 
Phosphate,  amorphous,  353 

sediments,  353 
Phosphates,   14 

determination  of,  335 
Phosphatides,  48 
Phospho-proteins,  54,  72 
Phosphorus  excretion,  304 

in  body,  8 
Physical  blood  tests,  204 
Physiological  chemistry  and  medicine,  5 

Physiological    chemistry,    scope    of,    2 

Phytoglobulins,  69 

Phytovitellins,  69 

Pigments  of  bile,  266 

Pioneer  investigators,  5 

Plants  and  animals,  3 

Plasma  of  muscle,  70,  278 

salted,  179 
Plasmon,  72 

Pleural  transudates,  233 
Poisons  and  liver,  258 

from  intestine,  162 
Polarimeter,  31 
Polar i scope,  30 
Polarization  tests,  30 
Polypeptides,  64,  85,    149 
Polysaccharides,  17,  32 
Pork,  composition,  93 
Potassium  in  body,  8 


Potassium  indoxyl  sulphate,   151 
Practical  urine  tests,  313 
Precipitation  by  salts,  55 

limits,  55 
Precipitins,  218 

Preparation  of  bile  acids,  265 
Preservatives  in  milk,  244 
Pressure,   osmotic,  204 
Primary  albumose,  82 

phosphates,  14 
Products  of  peptic  digestion,  140 
Prolamins,  74 
Proline,  62 

Prolonged  digestion,   142 
Propepsin,  108 
Propionic  acid,  40 
Protagon,  276 
Protalbumose,  82 
Protamines,  75 
Proteans,  69 
Proteids,  53,  85 
Protein  classification,  52 

combination,  64 

digestion,   128 

in   foods,  93,  94 

metabolism  theories  of,  290 

required,  378 
Proteins,  coagulating,  69 

determination,  59 

heats  of  combustion,  367 

in  autolysis,  257 

in  feces,  173 

in  urine,  305 

of  muscle,  278 

pancreatic  digestion,    146 

substances,  51 

synthesis,  64 
Proteolytic  reactions,  107 
Proteoses,  81 

in  feces,  173 

in  urine,  316 
Prothrombin,  178 
Protones,  75 
Protoplasm,  250 
Pseudo  acids,  56 

bases,  56 

cellulose,  39 

pepsin,  156 
Psychic  stimulus,  126 
Ptyalin,  97,  124 
Purine,  298 

bodies,  297 
Purines,  87 


392 


INDEX. 


Purines,  determination  in  urine,  333 
Pus,  232 

in  urine,  343 
Pyrimidine  bodies,  300 
Pyrimidines,  87 
Pyrrolidine  carboxylic  acid,  62 

Quadriurates,  299 

Quantitative  composition  of  blood,   176 

spectrum  analysis,  197 
Quotient,  respiratory,  356 

Raffinose,  27 

Ratios,  isodynamic,  374 

Reaction,  diazo,  326 

of  blood,  180 

of  feces,  166 
Reactions,  ferments,  97 

of  fats,  41 

proteins,  54 
Receptors,  226 
Reduced  hemoglobin,  195 
Reducing  power  of  sugars,  28 

of  urine,  307 
Reduction  tests,  22 
urine,  318 
Rennet,    108 

action  on  milk,  242 
Rennin,  72,  108,  126 
Reproductive  glands,  275 
Required  protein,  378 
Resorcinol  test,  24 
Respiration  apparatus,  357 

experiments,  358 

gases  of,  13 
Respiration  in  plants,  3 

skin,  363 
Respiratory  quotient,  356 
illustrations,  359 
Reversible  reactions  with  proteins,  77 
Ricinoleic  acid,  41 
Riegel  test  meal,  133 
Rotation,  specific,  32 

Saccharic  acid,  18 
Saccharodioses,    17 
Saccharomycetes,  112 
Saccharose,  25 
Saccharotrioses,  17,  27 
Salicin,  107 
Saliva,  121 
Salivary  diastase,  97 
digestion,  121 


Salkowski's  test  for  cholesterol,  50 

Salmin,  75 

Salmo-histone,  75 

Salted  plasma,  179 

Salt,  need  of,  16 

Salts,  and  proteins,  56 

in  body,  14 

in  blood,  177,  190 

of  casein,  72 

of  milk,  240 

of  muscle,  282 
Saponification,  41 
Sarcolactic  acid,  281 
Sauerkraut,  acid  in,  118 
Scheele,  97 
Schuetzenberger,  5 
Schultz  prism,  200 
Schweitzer's  reagent,  39 
Scomber  histone,  75 
Scombrin,  75 

Scope  of  physiological  chemistry,  2 
Secondary  albumose,  82 

phosphates,  14 
Sediments  from  urine,  306,  340 
Self  preservation  of  blood,  216 

purification  of  water,  9 

regeneration  of  cells,  227 
Semipermeable  membrane,  205 
Serine,  62 
Serum  albumin,  66 

globulin,  66,  68 

immunity,  227 

pus,  233 

tests,  219 
Side  chain  theory,  225 
Silicon  in  body,  8 
Silver  nitrate,  uses  of,  338 

test,  10 
Simple  proteins,  54 
Sizes  of  starch  grains,  34 
Skatol,  157,  160 
Skimmed  milk,   242 
Skin,  ash  of,   14 
Skin  respiration,  363 
Soap  and  hard  water,  42 
Soaps  in  feces,  168 
Sodium  in  body,  8 
Soft  water,  8 
Solids  in  feces,  167 

of  blood,  177 

of  body,  7 

of  milk,  243 
Solubility  of  fats,  44 


INDEX. 


393 


Soluble  ferments,  99 

starch,  33 
Sorbinose,  25 
Soxhlet  extraction   of   fat,  244 

sugar  values,  28 
Special  diets,  377 
Specific  gravity  of  urine,  313 
Specific  rotation,  31 

of  arabinose,  20 
of  bile  acids,  266 
of  cholesterol,  271 
of  dextrins,  38 
of  edestin,  69 
of  egg  albumin,  67 
of  fibrinogen,  70 
of  fructose,  24,  32 
of  glucose,  24,  32 
of  hemoglobin,  182 
of  invert  sugar,  32 
of  lactic  acid,  282 
of  lactose,  26,  32 
of  maltose,  27,  32 
of  melitose,  27,  32 
of  saccharose,  26,  32 
of  serum  albumin,  66 
of  serum  globulin,  69 
of  xylose,  20 
Spectroscope,   193 
Spectrum  analysis,  197 
of  blood,   193 

of  carbon  monoxide  hemoglobin,  197 
of  methemoglobin,  197 
of  oxyhemoglobin,  195 
of  reduced  hemoglobin,  195 
Spermaceti,  49 
Spermatozoa  in  urine,  347 
Spermine,  276 
Spleen,  234 

ash  of,  14 
Splitting  of  fats,  107 
Starch  digestion,  122 
Starches,  33 
Starch  in  feces,  170 

su^ar,  21 
Steapsin,  107,  154 
Stearic  acid,  40 
Stearin,  45 
Stercorin,  271 
Stomach,  acids  in,  134 
actions  in,  126 
contents,  tests,  132 
Sturin,  75 
Substratum,   ferment,    101 


Sucrase,  105 

Sudan  III  reagent,  48 

Sugar  from  malt,  123 

in  feces,  171 

in  milk,  240 

of  blood,  189 

of  malt,  26 
Sugars,   17 

determination,  27 

heats  of  combustion,  367 

in  urine,  304 

determination,  320 

reducing,  22 

relations  of,  18 

synthesis  of,  19 

tests  for  in  urine,  317 
Sulphates,  ethereal,  261 

in  body,  16 

in  urine,  303 
Sulpho  hemoglobin,  186 
Sulphur  compounds  from  proteins,  63 

distribution   of   in   urine,  303 

excretion,  302 
Sulphur  in  body,  8,   16 

in  keratin,  92 

neutral,  262,  303 
Supply  of  blood,  175 
Suprarenin,  273 
Suprarenal  bodies,  273 
Swedish  filter  paper,  38 
Symbiotic  processes,  118 
Syntheses   in  liver,  259 
Synthesis  of  ethereal  sulphates,  261 

of  polypeptides,   149 

of  sugar,   19 

of  uric  acid,  260 
Syntonin,  79 
Syrup,  glucose,  22 

Table  of  body  elements,  8 
Tallow,  46 

crystals,  43 
Tallquist  chart,  203 
Talosc,  24 
Tartaric  acid,   18 
Tartronic  acid,  18 
Taurin,  266 
Taurocholic  acid,  264 
Temporary  hardness,  9 
Tendons,  mucoids  in,  89 
Tension,  osmotic,  209 
Tertiary  phosphates,  14 
Test,  Almen's,  327 


394 


INDEX. 


Test,  biuret,  57 
bismuth,  319 
Boas',   134 
congo  red,  133 

dimethylaminoazobenzene,  133 
Donne's,  343 
double  iodide,  314 
Fehling's,  22 

for  urine,  318 
ferric  chloride,  323 
Gmelin's,  267 
guaiacum,  180 
Guenzberg's,  133 
Hammarsten's,  268 
Heller's,  326 
hydrogen  peroxide,  180 
Kelling's,  135 
lead  hydroxide,  58 
Legal's,  322 
Lieben's,  322 
methyl-violet,  133 
Moore's,  317 
murexide,  331 
phenylhydrazine,  319 
picric  acid,  314 
Struve's,  326 
Tanret,  314 
Trommer's,  318 
Trousseau's,  325 
Uffelmann's,  135 
xanthoproteic,  58 
Test  meals,  132 
Tests  for  acetoacetic  acid,  322 
acetone,  322 
air,  13 
albumins,  54  to  59 

in  urine,  314 
alcohol,   113 
ammonia  in  water,  10 
bile  colors,  267 
bile  salts,  266 
blood,  180 

in  urine,  326 
chlorides,    10 
cholesterol,  50 
colors  in  urine,  324 
creatinine,  334 
digestive  products,  131 
drinking  water,  9 
fats,  42,  47 

in  flour,  94 

in  milk,  242 
free  acid  in  stomach,  133 


Tests  for  gelatin,  91 

globulins  in  urine,  315 

glycogen,  36 

hemoglobin,  195 

indol,  152 

lactic  acid,   135 

leucine,  148 

levulose,  24 

meat  extract,  285 

milk  constituents,  242 

mucin  in  urine,   316 

muscle  extractives,  79 

nitrates,  11 

nitrites,  11 

organic  acids  in  stomach,  134 
nitrogen,  54 

oxybutyric  acid,  322 

pentose,  20 

pepsin,  139 

proteins,  54 

in  feces,  173 

proteoses,  131 

saliva,  121 

starch,  34 

sugar,  22 

in  milk,  242 
in  urine,  317 

sulphur  in  proteins,  65 

thiocyanates  in   saliva,   121 

tryptophane,  147 

tyrosine,  148 

urea  in  urine,  328 

uric  acid  in  urine,  331 
Tests  on  blood,   179 
bones,  287 
calculi,  354 
pepsin,  331 
Theories  of  fermentation,  98 

indicators,   136 

side  chain,  225 
Thiocyanates  in  saliva,   121 
Thrombin,  178 
Thymine,  187 
Thymus  cells,  233 
Thyreoglobulin,  274 
Thyroid  gland,  274 
Thyroiodine,  275 

Time  and  place  of  oxidation,  364 
Time  of  coagulation,  180 
Tissue  oxidation,  364 
Tissues,   ash  in,   14 

water  in,  12 
Titration  of  gastric  juice,  138 


INDEX. 


395 


Titration  of  stomach  contents,  132 
Total  fat  in  feces,  168 

hydrochloric  acid  in  stomach,  134 

nitrogen  in  urine,  327 
Toxins,  227 

from  intestine,  162 
Toxoids,  228 
Toxons,  228 

Transformation  products,  53,   76 
Transfusion  of  blood,  192 
Transudations,  232 
Treatment,   opsonic,  223 
Trehalose,  27 
Triolein,  45 
Triose,  18 

Trioxyglutaric  acid,  18 
Tripalmitin,  45 
Triple  phosphate,  304 
Trisaccharides,  17,  27 
Tristearin,  45 
Trommer  test,  22 
Trousseau's  test,  325 
True  albumins,  53,  65 
Trypsin,  83,  no,  145 

antipeptone,  150 
Trypsinogen,  145 
Tryptophane,   63,    147 
Turanose,  27 
Turkey,  composition,  93 
Tyrosine,  63,  92,  116 

in  urine,  350 

group,  56 

tests  for,  147 
Tyrosinase,   116 

Uffelmann's  test,  135 
Unit  of  force,  367 

of  heat,  366 

of  work,  367 
Unorganized   ferments,  99 

sediments,  341 
Uracil,  87 

Uranium  solution,  uses,  336 
Urates,  299 

in  sediment,  350 
Urea,  295 

decomposition,  296 

rmination,  328,  329 

fermentation,  11 1 

found  in  liver,  259 

synthesis,  296 
Urease,  11 1 
Uric  acid,  297,  331 


Uric  acid,  calculi,  355 

determination,  332 

from  spleen,  234 

in  liver,  260 

reducing  power,  308 

sediment,  349 
Urinary  calculi,  354 
Urine,  acetoacetic   acid  in,  322 
actone   in,   322 
albumin  in,  314 
ammonia  in,  330 
analysis,  313 
bacteria  in,  348 
blood  in,  326,  342 
calculi  from,  354 
casts,  345 
chlorides  in,  22>7 
color  and  odor,  292 
coloring  matters  in,  323 
conductivity,  309 
creatinine,  300 
cryoscopy,  312 
epithelium,  343 
fat  globules  in,  351 
fermentation,  in 
freezing  point,  312 
fungi  in,  347 
general  composition,  291 
leucine  and  tyrosine,  351 
molds  in,  348 
mucin,  316 

bands,  344 
mucus  in,  342 

nitrogen  compounds  in,  294 
oxalate  sediment,  352 
oxybutyric   acid   in,  322 
peptones  in,  316 
phosphate  in,  335 

sediment,  353 
proteins  in,  305 
proteoses  in,  316 
purines  in,  298 
pus  in,  343 
reaction,  293 
reducing  power,  307 
sediments,  306,  340 
spermatozoa  in,  347 
sulphates  in,  339 
total  nitrogen,  327 

sulphur,  3yj 
triple  phosphate  in,  353 
urates  in,  350 
urea  in,  295 


396 


INDEX. 


Urine,  uric  acid  in,  297 

sediment,  349 
xanthine  bodies  in,  298 
Urobilin,  324 
Urochrome,  324 
Uroerythrin,  324 
Urohematin,  324 
Urophain,  324 
Uses  of  chlorine  in  body,  15 

Value  of  blood  conductivity,  214 
Variations  in  blood,  191 
Vegetables,  composition,  94 
Vegetable  proteins,   73 
Vinegar,  115 
Vitellin,  73 

Vitreous  body,  mucoid  in,  89 
Voit,  5 

theory  of  protein  metabolism,  290 
Volhard's  method,  338 

Water,  distillation,  9 

in  body,  8 

in  tissue,  12 

physiological   importance,    12 

purification,  9 

tests,  9 
Waxes,  49 


Waxy  casts,  347 
Wheat  flour,  73 

starch,  S3 
Wheatstone  bridge,  213 
Whey,  240 

sugar    from,   26 
White  of  egg,  67 
Widal   test,   222 
Wood  paper,  38 

sugar,  20 
Wool  fat,  49 
Works  of  Liebig,  4 
Wohler,  4 
Wright's  coagulometer,  180 

Xanthine,  87 

bodies,   280 

in  urine,  298 

calculi,  355 
Xanthoproteic  test,  58 
Xylose,  20 

Yeast,   112 

action  of,  95 

Zymase,  99,  114 
Zymotoxic  group,  228 


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